Methods of high-throughput screening for internalizing antibodies

ABSTRACT

This invention provides methods of identifying ligands that are internalized into a cell. The methods typically involve i) contacting the cell with a reporter non-covalently coupled to a ligand; ii) dissociating the reporter from the ligand and removing dissociated reporter from the surface of the cell; and iii) detecting the reporter within said cell (if any is present) where the presence of the reporter within said cell indicates that the ligand binds to an internalizing receptor and is internalized.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/981,636 filed Oct. 16, 2001, which application claimsbenefit of and priority to U.S. Ser. No. 60/241,279, filed on Oct. 18,2000, the disclosures of which are incorporated herein by reference intheir entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported, in part, by Department of Defense Breast CancerResearch Program Grant Nos: DAMD17-94-J-4433 and DAMD17-98-1-8189. TheGovernment of the United States of America may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

With substantial completion of the first human genome sequencingproject, considerable attention has turned to a determination of thebiologic function of various DNA sequences. This investigation, oftentermed “functional genomics” represents a new phase of genome analysis.Specifically, functional genomics refers to the development andapplication of global (genome-wide or system-wide) experimentalapproaches to assess gene function by making use of the information andreagents provided by structural genomics. It is typically characterizedby high throughput or large-scale experimental methodologies combinedwith statistical and computational analysis of the results.

One fundamental strategy in a functional genomics approach is to expandthe scope of biological investigation from studying single genes orproteins to studying all genes or proteins at once in a systematicfashion. Computational biology will perform a critical and expandingrole in this area: whereas structural genomics has been characterized bydata management, functional genomics will be characterized by mining thedata sets for particularly valuable information. Functional genomicspromises to rapidly narrow the gap between sequence and function and toyield new insights into the behavior of biological systems.

One important class of genes includes those genes that encode cellsurface molecules and receptors. Receptors typically bind ligandsresulting in the delivery of a signal into the cell (signaling). Thiscan lead to a number of biologic functions including, but not limited tocell growth, cell replication, cell death, etc. Other receptors mediatethe specific transfer of molecules from outside the cell into thecytoplasm (endocytosis or internalization). Endocytosis is also animportant mechanism by which receptor signaling is modulated. Differentcell types have qualitatively and quantitatively different surfacereceptors and the pattern of receptor expression may change dramaticallywith the development and/or differentiation of a cell or tissue and/orthe development and progression of a disease.

Identification of such receptors and the development of specificreceptor ligands, allows the study of receptor function and thedetermination of the temporal-spatial pattern of receptor expression.For example, such ligands can be used to profile the pattern of receptorexpression across different cell types upon exposure to a drug or duringthe development of a disease. In addition, cell-specific receptorligand, more preferably internalizing cell specific receptor ligands canbe used to target drugs or markers to the cell surface or into thecytoplasm (for internalizing receptors), e.g. for therapeutic effect.

SUMMARY OF THE INVENTION

This invention provides methods for identifying cell binding andinternalizing ligands. Also provided are methods of identifyingreceptors that are capable of internalizing ligands and methods ofscreening for modulators of ligand internalization.

In one embodiment this invention provides a method of identifying aligand or ligands that are internalized into a cell. The method involvesi) contacting the cell with an effector (e.g. a reporter) non-covalentlycoupled to a ligand; ii) dissociating the reporter from the ligand andremoving dissociated reporter from the surface of the cell; and iii)detecting the reporter within the cell, if the reporter is presentwithin the cell, where the presence of the reporter within the cellindicates that the ligand binds to an internalizing receptor and isinternalized. In certain embodiment the contacting comprises contactingthe cell with a ligand comprising an epitope tag and contacting the cellwith a reporter comprising a moiety that binds the epitope tag. In apreferred embodiment the ligand is a ligand that binds to a cell surfacereceptor. Preferred ligands include, but are not limited to peptides(e.g. an scFv, an Fv, an Fab, monoclonal antibody, a cytokine, achemokine, a growth factor, etc.), nucleic acids, carbohydrates, sugars,and the like. Particularly preferred peptide ligands are produced bycombinatorial chemical synthesis or recombinantly using a phage displaylibrary (e.g. using a filamentous phage).

In certain preferred embodiments, the effector (e.g. reporter) isnon-covalently coupled to the ligand by an epitope tag (e.g. a His-tag,a Flag-tag, an HA-tag, a myc-tag, a DYKDDDDK (SEQ ID NO: 1) epitope,etc.). Where the effector is a reporter, preferred reporters include,but are not limited to an enzyme, a colorimetric label, a fluorescentlabel, a luminescent label, a radioactive label, a liposome, or aliposome containing a label. In one particularly preferred embodimentthe epitope tag is a hexahistidine (His₆) tag and said reporter is aliposome comprising a reagent that binds a His₆ tag (e.g.nitrilotriacetic acid (NTA)) attached to a lipid or liposome. In certainparticularly preferred embodiments, the attachment is typically via ametal chelation bond, e.g. a Ni(2+) chelation bond. In another preferredembodiment the ligand is an antibody and said epitope tag is attached tothe antibody through a covalent linkage to protein A.

Preferred cells for use in the methods of this invention include, butare not limited to plant cells, animal cells, and bacterial cells.Particularly preferred cells include mammalian cells, more preferablynormal or pathological human cells (e.g. a cancer cell). In certainembodiments the cells are cells that overexpress one or more receptorsand/or that express or overexpress a heterologous receptor.

The method can further involve isolating a ligand that is internalizedinto the cell. In certain embodiments, the “isolating” can comprisedetermining the amino acid sequence of a ligand that is internalized bythe cell or determining the sequence of a nucleic acid encoding theligand.

In another embodiment, this invention provides methods of screening acell for internalization of a ligand. These methods preferably involvei) contacting the cell with a reporter non-covalently coupled to aligand known to be internalizing; ii) dissociating the reporter from theligand and removing dissociated reporter from the surface of the cell;iii) detecting the reporter within said cell, if said reporter ispresent within said cell, whereby the presence of the reporter withinsaid cell indicates that said cell internalizes said ligand. Mostfrequently, internalization of a ligand into a cell signifies that thecell displays a receptor for the ligand that is an internalizingreceptor. The method may further include isolation of the cell thatinternalized the ligand, e.g. from those cells that do not.

In a particularly preferred embodiment the ligand is a member of alibrary of ligands. Preferred libraries comprise at least 1000, morepreferably at least 10,000, and most preferably at least 100,000different members. Preferred ligands include, but are not limited topeptides (e.g. an scFv, an Fv, an Fab, monoclonal antibody, a cytokine,a chemokine, a growth factor, etc.), nucleic acids, carbohydrates,sugars, and the like. Particularly preferred peptide ligands areproduced by combinatorial chemical synthesis or recombinantly using aphage display library (e.g. using a filamentous phage).

In certain preferred embodiments, the effector (e.g. reporter) isnon-covalently coupled to the ligand by an epitope tag (e.g. a His-tag,a Flag-tag, an HA-tag, a myc-tag, a DYKDDDDK (SEQ ID NO:1) epitope,etc.). Where the effector is a reporter, preferred reporters include,but are not limited to an enzyme, a colorimetric label, a fluorescentlabel, a luminescent label, a radioactive label, a liposome, or aliposome containing a label. In one particularly preferred embodimentthe epitope tag is a hexahistidine (His₆) tag and said reporter is aliposome comprising a reagent that binds a His₆ tag (e.g.nitrilotriacetic acid (NTA)) attached to a lipid or liposome. In anotherpreferred embodiment the ligand is an antibody and said epitope tag isattached to the antibody through a covalent linkage to protein A.Particularly preferred cells are described herein.

In certain embodiments, the method further comprises isolating a ligandthat is internalized into the cell. The ligand can be sequenced or thesequence of a nucleic acid encoding the ligand is determined. The methodmay further comprise contacting a cell with a labeled ligand again totag or isolate the internalizing receptor.

In yet another embodiment, this invention provides methods ofidentifying internalizing receptors. The methods involve i) contacting acell with a reporter non-covalently coupled to a ligand; ii)dissociating the reporter from the ligand and removing dissociatedreporter from the surface of said cell; iii) detecting the reporterwithin said cell, if said reporter is present within said cell, wherebythe presence of the reporter within said cell indicates that said ligandbinds to an internalizing receptor and is internalized; iv) identifyingor recovering the ligand bound to the reporter within said cell; and v)identifying a receptor that binds to the ligand. In particularlypreferred embodiments, the receptor is identified by methods including,but not limited to affinity chromatography or immunohistochemistry. Themethod can further comprise entering the identity of the internalizingreceptor into a database of internalizing receptors.

Also provided are methods of method of screening an agent for theability to modulate internalization of a ligand into a cell. The methodspreferably involve i) contacting the cell with a reporter non-covalentlycoupled to a ligand known to be internalized by said cell; ii)contacting the cell with a test agent; iii) dissociating the reporterfrom the ligand and removing dissociated reporter from the surface ofthe cell; and iv) detecting the reporter within the cell, if thereporter is present within the cell, where a difference in the amount ofreporter internalized by the cell contacted with said test agent ascompared to the amount of reporter internalized by said cell whencontacted with a lower concentration of the test agent indicates thatsaid test agent modulates the internalization of said ligand by thecell. In preferred embodiments, the lower concentration of test agent isthe absence of the test agent. Preferred test agents include smallorganic molecules. In certain embodiments, the test agents includeantibodies or peptides while in certain embodiments, the test agents donot include nucleic acids, antibodies, or peptides.

In still another embodiment, the method involves contacting the cellwith a first concentration of the test agent; ii) contacting the cellwith a reporter non-covalently coupled to a ligand known to internalizeinto the cell; iii) dissociating the reporter from the ligand andremoving dissociated reporter from the surface of the cell; iv)detecting the reporter within the cell to obtain a first measurementthat signifies the amount of the reporter/ligand construct internalizedby the cell; v) contacting said cell with a second concentration of thetest agent wherein the second concentration is higher that the firstconcentration; vi) repeating the steps ii)-iv) to obtain a secondmeasurement that signifies the amount of the reporter/ligand constructinternalized by the cell influenced by a second, higher concentration ofthe agent; and vii) comparing the first and the second measurementswherein when the first and the second measurements are different, thetest agent modulates internalization of said ligand in said cell.

In certain preferred embodiments, the first, lower concentration of testagent is zero, i.e. the absence of the test agent. Preferred test agentsinclude small organic molecules. In certain embodiments, the test agentsinclude antibodies or peptides while, in certain embodiments, the testagents do not include nucleic acids, antibodies, or peptides.

In still another embodiment, this invention provides a construct for usein the methods of this invention (e.g. for screening a cell for aninternalizing receptor). Preferred constructs comprise a ligandnon-covalently coupled to an effector (e.g. a reporter) through anepitope tag. In preferred constructs the ligands include, but are notlimited to peptides (e.g. an scFv, an Fv, an Fab, monoclonal antibody, acytokine, a chemokine, a growth factor, etc.), nucleic acids,carbohydrates, sugars, and the like. Particularly preferred peptideligands are produced by combinatorial chemical synthesis orrecombinantly using a phage display library (e.g. using a filamentousphage).

In certain preferred constructs, the effector (e.g. reporter) isnon-covalently coupled to the ligand by an epitope tag such as aHis-tag, a Flag-tag, an HA-tag, a myc-tag, a DYKDDDDK (SEQ ID NO:1)epitope, etc. Where the effector is a reporter, preferred reportersinclude, but are not limited to an enzyme, a colorimetric label, afluorescent label, a luminescent label, a radioactive label, a liposome,or a liposome containing a label. In one particularly preferredembodiment the epitope tag is a hexahistidine (His₆) tag and thereporter is a liposome comprising a reagent that binds a His₆ tag (e.g.nitrilotriacetic acid (NTA)) attached to a lipid or liposome. In anotherpreferred embodiment the ligand is an antibody and said epitope tag isattached to the antibody through a covalent linkage to protein A. Incertain preferred embodiments, the construct is polyvalent for theligand.

This invention also provides ligand libraries for use in the methods ofthis invention. Preferred libraries comprise a plurality of constructsas described herein where the members of the library each comprise aligand and an epitope tag where the ligands vary between members of thelibrary and the epitope tags are constant among members of the library.The ligand/effector (e.g. reporter) components of the library membersmay be pre-assembled or may assemble during when they are combined, e.g.in the presence of a cell. Preferred libraries comprise at least 10⁵different ligands.

In still another embodiment this invention provides a kit for screeninga cell for an internalizing receptor. Preferred kits comprise aconstruct or a library of constructs as described herein. Preferred kitsfurther comprise instructional materials teaching the use of saidlibrary to screen for internalizing ligands or to identify aninternalizing receptor.

In yet another embodiment, the invention provides method of detectingbinding and internalization of the ligands by cells. The method involvesi) contacting the cell with an effector (e.g. a reporter) non-covalentlycoupled to a ligand; ii) removing a portion of the effector which is notassociated with the cell; iii) detecting the reporter associated withthe cell to obtain a first reading indicating a total amount of theligand which is bound to the cell surface and internalized by the cell;iv) dissociating the reporter from the ligand and removing dissociatedreporter from the surface of the cell; v) detecting the reporterremaining in the cell to obtain a second reading indicating an amount ofthe ligand which is internalized; and vi) subtracting the second readingfrom the first reading to obtain a difference indicating an amount ofthe ligand bound to cell surface. In some cases, following thecontacting step it is advantageous to arrest further internalizationprocess, for example, by reducing temperature of the cells, typically toabout 4° C., or by treatment of the cells with effective amounts ofmetabolic inhibitors, e.g. anhydroglucose or sodium azide. In certainembodiment the contacting comprises contacting the cell with a ligandcomprising an epitope tag and contacting the cell with a reportercomprising a moiety that binds the epitope tag. In a preferredembodiment the ligand is a ligand that binds to a cell surface receptor.Preferred ligands include, but are not limited to peptides (e.g. anscFv, an Fv, an Fab, monoclonal antibody, a cytokine, a chemokine, agrowth factor, etc.), nucleic acids, carbohydrates, sugars, and thelike. Particularly preferred peptide ligands are produced bycombinatorial chemical synthesis or recombinantly using a phage displaylibrary (e.g. using a filamentous phage).

In certain preferred embodiments, the effector (e.g. reporter) isnon-covalently coupled to the ligand by an epitope tag (e.g. a His-tag,a Flag-tag, an HA-tag, a myc-tag, a DYKDDDDK (SEQ ID NO:1) epitope,etc.). Where the effector is a reporter, preferred reporters include,but are not limited to an enzyme, a colorimetric label, a fluorescentlabel, a luminescent label, a radioactive label, a liposome, or aliposome containing a label. In one particularly preferred embodimentthe epitope tag is a hexahistidine (His₆) tag and said reporter is aliposome comprising a reagent that binds a His₆ tag (e.g.nitrilotriacetic acid (NTA)) attached to a lipid or liposome, e.g. via ametal chelation bond, such as Ni(2+) chelation bond. In anotherpreferred embodiment the ligand is an antibody and said epitope tag isattached to the antibody through a covalent linkage to protein A orprotein G.

Preferred cells for use in the methods of this invention include, butare not limited to, plant cells, animal cells, and bacterial cells.Particularly preferred cells include mammalian cells, more preferablynormal or pathological human cells (e.g. a cancer cell). In certainembodiments the cells are cells that overexpress one or more receptorsand/or that express or overexpress a heterologous receptor.

The invention also provides for metal-chelating lipids comprisingsterols and capable of forming metal chelation bond with an epitope tag,preferably, with a hexahistidine tag. More preferably, metal-chelatinglipids containing cholesterol-conjugated NTA metal complex are provided.

The invention also provides for metal-chelating lipids comprising alipid, a hydrophilic polymer, and a chelation group attached to saidhydrophilic polymer Preferably, the invention provides for thepoly(ethylene glycol)-lipid conjugates containing a terminally attachedmetal chelation group. More preferably, the conjugates comprising apoly(ethylene glycol)-lipid and a terminally attached metal chelationgroup capable of forming a chelation bond with an epitope tag, such asan oligohistidine tag, are provided. In a particular embodiment,poly(ethylene glycol)-lipid is poly(ethylene glycol)-conjugated DSPE,and a chelation group is NTA.

The invention also provides for compositions comprising metal chelatinglipids comprising a lipid, a hydrophilic polymer, and a chelation groupattached to said hydrophilic polymer and capable of forming a chelationbond with an epitope tag. The invention further provides for the methodsfor delivery of an effector into a cell comprising contacting the cellwith (i) a metal chelating lipid comprising a lipid, a hydrophilicpolymer, and a chelation group attached to said hydrophilic polymer andcapable of forming a chelation bond with an epitope tag, wherein saideffector is associated with said metal chelating lipid, and (ii) aligand comprising said epitope tag wherein said cell specifically binds,and optionally, internalizes, said ligand. The composition preferablyincludes a liposome, which comprises said metal chelating lipid and saideffector.

DEFINITIONS

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The term also includes variants on the traditionalpeptide linkage joining the amino acids making up the polypeptide.Proteins also include glycoproteins (e.g. histidine-rich glycoprotein(HRG), Lewis Y antigen (Le^(Y)), and the like.).

The terms “nucleic acid”, or “oligonucleotide” or grammaticalequivalents herein refer to at least two nucleotides covalently linkedtogether. Nucleic acids of the present invention are single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111 :2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Letsinger et al. (1988)J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside &Nucleotide 13:1597; Chapters 2 and 3, ACS Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem.Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; TetrahedronLett. 37:743 (1996)) and non-ribose backbones, including those describedin U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ACSSymposium Series 580, Carbohydrate Modifications in Antisense Research,Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of additional moieties such as labels,or to increase the stability and half-life of such molecules inphysiological environments.

The term “residue” as used herein refers to natural, synthetic, ormodified amino acids.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (sFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H)-V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv′s (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No: 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and4,956,778). Particularly preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

The term “specifically binds”, as used herein, when referring to abiomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to abinding reaction which is determinative of the presence biomolecule inheterogeneous population of molecules (e.g., proteins and otherbiologics). Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody or stringent hybridizationconditions in the case of a nucleic acid), the specified ligand orantibody binds to its particular “target” molecule and does not bind ina significant amount to other molecules present in the sample.

The term “ligand” refers to a molecule that is or that can bespecifically bound by and/or transported by another molecule. Preferredligands include, but are not limited to peptides, nucleic acids,carbohydrates, sugars, hormones, and the like. A ligand and a moleculethat it binds form a binding pair, in which each one member is regardedas a ligand in respect to the other member. Specific examples of bindingpairs include antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, transporter protein/substrate, lectin/carbohydrate,receptor/hormone, receptor/modulator, complementary strands ofpolynucleotides, protein/nucleic acid repressor(inductor),receptor/virus, etc.

The term “nanoparticle”, as used herein refers to a “vehicle” capable ofcomplexing with or containing an effector (e.g. a drug, a detectablelabel, a cytotoxin, etc.). A preferred nanoparticle also provides anon-covalent or cleavable covalent linkage to a ligand (direct orthrough a linker).

An “effector” refers to any molecule or combination of molecules whoseactivity it is desired to internalize into a cell. Effectors include,but are not limited to labels, cytotoxins, enzymes, growth factors,transcription factors, drugs, etc.).

A “reporter” is an effector that provides a detectable signal (e.g. is adetectable label). In certain embodiments, the reporter need not providethe detectable signal itself, but can simply provide a moiety thatsubsequently can bind to a detectable label.

The term “modulate” when used with reference to modulation ofinternalization of a ligand refers to an upregulation or downregulationof the total amount or ligand internalized and/or the rate ofinternalization. In certain embodiments, particularly where ligandefflux is not assayed or otherwise controlled for, modulation may occurby altering the rate of efflux of the ligand and reflect net rate or netamount of ligand incorporation by a cell.

The term “test agent” refers to any agent that is to be screened for adesired activity (e.g. the ability to modulate/alter internalization ofa ligand by a cell). The “test composition” can be any molecule ormixture of molecules, optionally in a suitable carrier. The term “testcell” refers to any cell to which methods of the present invention areapplied.

The term “small organic molecule” typically refers to molecules of asize comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size up to about 5000 Da, more preferably up to 2000 Da, and mostpreferably up to about 1000 Da.

The term “detect” refers to detection or quantitative determination.

The term “chelation bond” refers to a bond between an effector and aligand which involves an interaction between an electron pair donor anda coordination site on a metal ion leading to an attractive forcebetween the electron pair donor and the metal ion.

The term “liposome” refers to a nanoparticle that comprises aself-enclosed layer composed of an amphipathic lipid. The layertypically is a bilayer formed by molecules that comprise a hydrophobicportion and a hydrophilic portion wherein hydrophobic portions associatein an aqueous medium to form an internal part of the layer whereashydrophilic portions remain in contact with the medium. The layersurrounds and encloses an interior, which may contain, wholly orpartially, an aqueous phase, a solid, a gel, a gas phase, or anon-aqueous fluid. An effector, e.g. a reporter, may be contained withinthe interior of the liposome, in the lipid layer, or attached to theouter surface of the lipid layer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of Ni-NTA-lipid in the liposome on theliposome internalization into cells in the presence of acell-internalizing ligand having a His₆ epitope tag. Small unilamellarliposomes containing encapsulated fluorescent marker were formulatedwith lipid matrix containing 0.5 mol. %, (squares), 2 mol. % (diamonds),or 5 mol. % (circles) of an NTA-lipid (DOGS-NTA-Ni, Avanti Polar Lipids,Inc., Alabaster, Ala.), (measured as mol. % of liposome phospholipid)and tested for internalization into ErbB2-expressing SKBR3 tumor cellsusing 20 μg/mL of an anti-ErbB2 scFv antibody (F5) engineered to containa C-terminal (His)₆-tag. After four hours of internalization, cells werewashed with physiological saline containing 1 mM EDTA, lysed in base andthe fluorescence read in a microfluorimeter.

FIG. 2 illustrates the specificity of the CLIA assay. SKBR3 tumor cellswere incubated with NTA-liposomes (5 mol. % Ni-NTA-DOGS) and theanti-ErbB2 antibody F5 without a (His)₆-tag, or a non-internalizinganti-ErbB2 antibody (C6.5), or no scFv. Alternatively, the F5 scFvcontaining the (His)₆-tag was co-incubated with fluorescently labeledliposomes formulated without the NTA-DOGS lipid. After four hours ofinternalization, cells were washed with physiological buffered salinecontaining 1 mM EDTA, lysed in base and the fluorescence read in amicrofluorimeter.

FIG. 31 illustrates monitoring of internalization monoclonal antibodiesby CLIA using a Protein A-(His)₆ chemical conjugate. SKBR3 cells wereincubated the anti-ErbB2 antibody F5 with a (His)₆-tag, theanti-ErbB2monoclonal antibody Herceptin, Protein A-(His)₆ alone, ormixture of Herceptin and Protein A-(His)₆. After four hours ofinternalization, cells were lysed in base and the fluorescence read in amicrofluorimeter.

FIG. 4 illustrates the effect of liposome concentration in the CLIAassay. SKBR3 cells were co-incubated with (squares) or without (circles)20 micro-g/mL of the anti-ErbB2 antibody F5 and varying concentrationsof Ni-NTA-liposomes with encapsulated fluorescent marker. After fourhours of internalization, cells were washed with physiological salinebuffer containing 1 mM EDTA, lysed in base and the amount ofcell-associated liposome lipid was determined from the fluorescence readin a microfluorimeter.

FIG. 5 illustrates the effect of antibody concentration in the CLIAassay. SKBR3 cells were co-incubated with varying concentrations of theanti-ErbB2 antibody F5 (solid line-circles), a control antibody(squares), or no antibody (dotted line-circles) and NTA-liposomescontaining 2 mol. % Ni-NTA-DOGS. After four hours of internalization,cells were washed with physiological saline buffer containing 1 mM EDTA,lysed in base and the fluorescence read in a microfluorimeter.

FIG. 6 shows the use of culture supernatants in the CLIA assay. SKBR3cells were co-incubated with culture supernatants of E. coli expressingthe anti-EGFR scFv antibody C10, the non-internalizing scFv antibodyC6.5, the anti-ErbB2 scFv antibody F5, or no scFv along withNTA-liposomes. After four hours of internalization, cells were washedwith physiological saline buffer containing 1 mM EDTA, lysed in base andthe fluorescence read in a microfluorimeter.

FIG. 7 illustrates tumor cell profiling with the anti-EGFR scFvantibody. The anti-EGFR antibody C10 was co-incubated withfluorescent-labeled NTA-liposomes on cell lines expressing varyingamounts of EGFR: SKBR3, SKOV3, BT474, MCF7, MD-MBA 453, MD-MDA 468,CHO-EGFR, or CHO. Uptake was normalized to total cellular protein.

FIG. 8 shows a comparison of fluorescent anti-ErbB2 staining by flowcytometry, uptake of covalent liposomes, and the CLIA assay. Tumor cellswere incubated with the anti-ErbB2 scFv F5, detected with FITC-labeledanti-FLAG antibody, and fluorescence quantified by flow cytometry.Alternatively, live tumor cells were incubated with immunoliposomescontaining the F5 scFv covalently coupled to the liposome containingencapsulated fluorescent marker HPTS. The CLIA assay was performed byco-incubation of the F5 scFv and NTA-liposomes. Liposome fluorescencewas read in a microfluorimeter.

DETAILED DESCRIPTION

This invention provides methods of identifying ligands that areinternalized into cells or to identify internalizing receptors that arecapable of internalizing ligands into cells. The methods involvecoupling a ligand non-covalently (e.g. via an epitope tag) to ananoparticle containing an effector (e.g. reporter molecules, etc.)without the need for ligand purification. Since purification is notrequired, either before or after exposure of a test cell to the ligandcoupled to the “nanoparticle”, cell binding and internalization canreadily be assayed in a high throughput manner.

In general, the methods involve providing an effector (e.g. reporter)non-covalently coupled to a ligand (e.g. a ligand generated in acombinatorial library). The effector/ligand is contacted with a “test”cell, e.g. a cell that is to be assayed for the ability to internalizethe ligand. The effector/reporter is dissociated from the ligand and thedissociated reporter is removed from the surface of the cell. Inpreferred embodiments, the reporter/effector is detected within the celland the presence of the reporter/effector within the cell indicates thatthe ligand is internalized. Most frequently, the internalization of aligand signifies that the cell displays an internalizing receptor thatbinds the ligand. The methods can further include identifying and/orisolating the cells that internalized the ligand (and hence, theeffector), for example, for a diagnostic or therapeutic purpose, whereinthe cells are pathological, e.g. cancer cells, to find out if thesecells are present in a tissue or fluid specimen from a patient, such asblood urine, sputum, or tissue biopsy. In another example, geneticallyengineered cells that express an internalizing epitope on their surfaceas a result of DNA transfection, can be detected and isolated. Becausethe surface-attached effectors are dissociated and removed undercell-sparing conditions which preserve the cell integrity, isolatedcells can be maintained and propagated yielding useful clones of stabletransfectants.

In another embodiment, this invention provides methods of identifying aninternalizing receptor. In preferred embodiments, such methods involveidentifying internalized ligands, e.g., according to the methodsdescribed above. The internalized ligands are recovered from the celland/or identified. The recovered and/or identified ligand can then beused to identify the receptor that internalized that ligand (e.g. bylabeling the receptor in situ, by affinity purifying the receptor,etc.).

In still another embodiment, the methods of this invention can be usedto screen for agents that modulate the ability of a cell to internalizea ligand. In preferred embodiments, these methods entail screening forligand internalization as described herein where the cells are contactedbefore or during the time they are contacted with the effector/ligandconstruct and) with the test agent(s) to be screened. A difference inligand internalization by cells contacted with the test agent(s), e.g.as compared to negative controls comprising the test agent(s) at a lowerconcentration or the absence of the test agent(s), indicates that thetest agent(s) modulate (e.g. increase or decrease) internalization thesubject ligand(s).

The invention also provides the methods of detecting binding andinternalization of the ligands by cells. The methods involves i)contacting the cell with an effector (e.g. a reporter) non-covalentlycoupled to a ligand; ii) removing a portion of the effector which is notassociated with the cell; iii) detecting the reporter associated withthe cell to obtain a first measurement indicating a total amount of theligand which is bound to the cell surface and internalized by the cell;iv) dissociating the reporter from the ligand and removing dissociatedreporter from the surface of the cell; v) detecting the reporterremaining in the cell to obtain a second measurement indicating anamount of the ligand which is internalized; and vi) subtracting thesecond measurement from the first measurement to obtain a differenceindicating an amount of the ligand bound to cell surface.

Providing an Effector Non-Covalently Coupled to a Ligand.

In a preferred embodiment, the methods of this invention utilize aneffector (typically complexed with or localized in a “nanoparticle”)non-covalently attached to a ligand. In certain embodiments, an effectorcan be attached to a ligand by a cleavable covalent bond.

Ligands for Coupling to an Effector.

Virtually any ligand is suitable for used in the methods of thisinvention. While, in particularly preferred embodiments, the methods ofthis invention utilize peptides, it is also possible to use nucleicacids, sugars, various carbohydrates, lipids, and any of a variety oforganic molecules as ligands.

In certain embodiments, a single ligand can be used to identify cellsand/or receptors capable of internalizing that ligand. In otherembodiments, multiple ligands can be used to identify internalizingreceptors and/or ligands that can be internalized by a particular cell.In particularly preferred embodiments, the ligands are provided ascomponents of libraries comprising large numbers of different ligands,sometimes referred to as combinatorial libraries. Use of large ligandlibraries comprising numerous different ligands increases the likelihoodof identifying a ligand that is internalized by a particular cell.

Preferred libraries include at least 2, preferably at least 5, morepreferably at least 10, and most preferably at least 100, or at least1000 different ligands. Even larger libraries are possible and oftenpreferred. Such larger libraries include at least 10,000 differentligands, preferably at least 100,000 different ligands, or even at leastabout 1,000,000 or more ligands.

Methods of producing combinatorial peptide libraries are well known tothose of skill in the art. Such peptide libraries can be chemicallysynthesized or produced by expressing libraries of nucleic acids. Theinitial work in combinatorial library construction focused on chemicalpeptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res.37:487-493; Houghton et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;and Fodor et al. (1991) Science 251:767.

Methods of generating peptide libraries using recombinant DNAtechniques, however, are becoming quite common. Thus, for example, theuse of phage display libraries and the like permit the generation ofsingle chain antibody or other peptide ligand libraries. To express suchlarge libraries a polypeptide or an antibody fragment gene is insertedinto the gene encoding a phage surface protein (pIII) and thepolypeptide-pIII fusion protein is displayed on the phage surface(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991)Nucleic Acids Res. 19: 4133-4137). The gene can include an enzymecleavage site permitting separation of the peptide from the pIII proteinas desired.

The nucleic acid(s) encoding the protein can be highly degenerate in oneor more regions thereby providing a library of literally thousands ofpeptides. (see, e.g., U.S. Pat. Nos. 5,198,346, 5,096,815, 4,946,778,etc.). Libraries have been constructed comprising over 100,000 or evenover 1,000,000 different members (see, e.g., Yang and Craik (1998) J.Mol. Biol., 279: 1001-1011).

Phage-display methods are not the only approach to the generation ofpeptide libraries. To the contrary, it is possible to generate largepeptide libraries using vectors other than phage.

While in certain embodiments, the ligands utilized in this invention are“randomly” generated, in embodiments, can involve building variationaround a peptide “lead.” In this approach, one starts with a particularpeptide sequence, the lead, which may have been selected by some otherrandom peptide approach, such as the peptides on phage approach,discussed above. One then synthesizes in vitro (e.g., with an automatedDNA synthesizer) a family of oligonucleotides that is based on thecoding sequence of the lead peptide. Each member of the family varies toa particular degree from the original sequence. Sources of leads include(1) quasi-random peptides generated, e.g. in phage display libraries (2)small peptide encoding DNAs derived from the genes for the naturalligands; (3) shuffling of small peptide-encoding fragments to introducevariation (see, e.g., U.S. Pat. Nos: 6,132,970, 6,117,679, 3 6,096,548);(4) peptide leads from other sources of peptide diversity andcharacterization that involve the intracellular generation of peptidediversity and detection of peptide-protein interactions via thereconstitution of a viable transcriptional transactivator (see, Fieldand Song, (1989) Nature 340(6230): 245-246); and (6) diverse peptidesbuilt around a specific conformationally constrained molecular scaffold(see, e.g., Yang and Craik (1998) J. Mol. Biol., 279: 1001-1011.

Yet another approach for diversifying a selected random peptide vectorinvolves the mutagenesis of a pool, or subset, of recovered vectors.Recombinant host cells transformed with vectors identified by screeningare pooled and isolated. The vector DNA, or a portion of the vector DNA,is mutagenized by treating the cells with, e.g., nitrous acid, formicacid, hydrazine, or by use of a mutator strain such as mutD5 (see, e.g.,Schaaper (1988) Proc. Natl. Acad. Sci., USA, 85: 8126-8130). Thesetreatments produce a variety of mutations in the vector DNA. The segmentcontaining the sequence encoding the variable peptide can optionally beisolated by cutting with restriction nuclease(s) specific for sitesflanking the variable region and then recloned into undamaged vectorDNA. Alternatively, the mutagenized vectors can be used withoutrecloning of the mutagenized random peptide coding sequence.

One can also diversify a selected peptide by misincorporation ofnucleotide changes in the coding sequence for the peptide with thepolymerase chain reaction (PCR; see U.S. Pat. Nos. 4,683,202; 4,683,195;and 4,965,188) under low fidelity conditions. A protocol described inLeung et al. (1989) Technique 1: 11-15, utilizes altered ratios ofnucleotides and the addition of manganese ions to produce a significantmutation frequency.

One can also use extensive mutagenesis to generate a large number ofpeptides with a significant number of differences from the lead (as wellas generating peptides with few or no differences from the lead). Inanother approach, single amino acid substitutions in the peptide arefavored, and the goal is to find a number of single amino aciddifferences that either abolish or significantly improve binding. Forexample, one approach involves the synthesis of four mixtures ofnucleotides—each containing one of the four nucleotides at 85%, and eachof the other three nucleotides at 5% each. Thus, at each position duringsolid phase chemical synthesis there is an 85% chance that the “correct”nucleotide will be incorporated and a 15% chance that one of the otherthree nucleotides will be incorporated (a 5% chance for each). Thus, onaverage, if one synthesizes an oligonucleotide 100 bases long, then inan average molecule 85% of the nucleotide positions will be correct(that is, will match the lead sequence), and 15% of the positions willhave incorporated an incorrect nucleotide compared to the originalsequence. Depending on the misincorporation criteria that are selected,the resulting mixture of different oligonucleotides can be quite similarto the core starting sequence, for example by following a 97% 1%/1%/1%misincorporation strategy, or quite diverged, on average, from the leadsequence, for example by following a 55%/15%/15%/15% strategy.

The approaches described above are merely illustrative. Other approachesto the generation of peptide libraries are well known to those of skillin the art (see, e.g. U.S. Pat. No. 5,010,175, Furka (1991) Int. J.Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354:84-88, and the like).

The ligands used in the methods of this invention are not limited topeptide ligands. Virtually any ligand can be utilized as long as it iscapable of non-covalently associating the effector/reporter. Moreover,it is possible to derivatize ligands, e.g. with a particular peptideepitope so that the ligand can non-covalently associate with aparticular effector/reporter. Suitable non-peptide ligands include, butare not limited to nucleic acids (RNAs, or DNAs, or analogues thereof),sugars, carbohydrates, lipids, small organic molecules and the like.

The scope of combinatorial chemistry libraries has been expanded beyondpeptide synthesis. Polycarbamate and N-substituted glycine librarieshave been synthesized to produce libraries containing chemical entitiesthat are similar to peptides in structure, but possess enhancedproteolytic stability, absorption and pharmacokinetic properties. Cho etal. (1993) Science 261:1303-1305; Simon et al. (1992) Proc. Natl. Acad.Sci., USA, 89:9367-9371. Furthermore, benzodiazepine, pyrrolidine, anddiketopiperazine libraries have been synthesized, expandingcombinatorial chemistry to include heterocyclic entities. Bunin et al.(1992) J. Am. Chem. Soc. 114: 10997-10998; Murphy et al. (1995) J. Am.Chem. Soc. 117: 7029-7030; and Gordon et al. (1995) Biorg. MedicinalChem. Lett. 5:47-50.

Methods of chemical and/or biological synthesis, by combining a numberof chemical “building blocks”, as reagents can produce libraries ofenormous complexity and diversity. For example, one commentator hasobserved that the systematic, combinatorial mixing of 100interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

Known combinatorial chemical libraries include, but are not limited to,:peptoids (PCT Publication No WO 91/19735, Dec. 26, 1991), randombio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta- D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan18, page 33, isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones andmetathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos.5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No. 5,506,337,benzodiazepines 5,288,514, pyrimidinediones (see, e.g., U.S. Pat. No.6,025,371), and the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include, but are not limitedto, automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimicthe manual synthetic operations performed by a chemist and the Venture™platform, an ultra-high-throughput synthesizer that can run between 576and 9,600 simultaneous reactions from start to finish (see AdvancedChemTech, Inc. Louisville, Ky.)). Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

Effector/Nanoparticle Combinations.

In preferred embodiments, the ligand is non-covalently coupled to aneffector. The coupling can be direct or to a vehicle “carrying” theeffector (e.g., a nanoparticle). As used herein an effector refers toany molecule or combination of molecules whose activity it is desired tointernalize into a cell. Effectors include, but are not limited tomolecules such as labels, cytotoxins, enzymes, growth factors,transcription factors, nucleic acids, drugs, etc.). The drugsparticularly suitable as effectors are cytotoxic anticancer drugs.Examples of cytotoxic anticancer drugs are anthacyclines (e.g.,doxorubicin), vinca alkaloids (e.g., vincristine, vinblastin,vinorelbine), folate analogs (e.g., methotrexate, edatrexate),nucleotide analogs (e.g. arabinosylcytidine, azathymidine), platinumcomplexes (e.g., cisplatin, carboplatin), and alkylating agents (e.g.,nitrosourea, melphalan, cyclophosphamide).

In particularly preferred embodiments, the effector comprises adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by spectroscopic,photochemical, electrochemical, biochemical, immunochemical, magnetic,electrical, optical or chemical means. Useful labels in the presentinvention include biotin for staining with labeled streptavidinconjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g.,fluorescein, Texas Red, rhodamine, green fluorescent protein, and thelike, see, e.g., Haugland (1996), Handbook of Fluorescent Probes andResearch Chemicals, 6th Edition, Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horseradish peroxidase, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

A fluorescent label is preferred because it provides a very strongsignal with low background. It is also optically detectable at highresolution and sensitivity through a quick scanning procedure.Desirably, fluorescent labels should absorb light above about 300 nm,preferably about 350 nm, and more preferably above about 400 nm, usuallyemitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound dye can differ from theunbound dye. Therefore, when referring to the various wavelength rangesand characteristics of the dyes, it is intended to indicate the dyes asemployed and not the dye which is unconjugated and characterized in anarbitrary solvent.

Detectable signals can also be provided by chemiluminescent andbioluminescent sources. Chemiluminescent sources include a compoundwhich becomes electronically excited by a chemical reaction and can thenemit light which serves as the detectable signal or donates energy to afluorescent acceptor. Alternatively, luciferins can be used inconjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electronspin which can be detected by electron spin resonance (ESR)spectroscopy. Exemplary spin labels include organic free radicals,transitional metal complexes, particularly vanadium, copper, iron, andmanganese, and the like. Exemplary spin labels include nitroxide freeradicals.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

Other preferred labels include radioactive labels. Radioactive labelsmay be introduced into a nanoparticle which then is non-covalentlylinked to an effector. For example, the isotopes of ¹²⁵I, ¹³¹I,^(99m)Tc, ⁶⁷Ga, ¹¹¹In, ¹⁴C, ³H, ³⁵C, and ¹⁴P are commonly used asradioactive labels. Radioactive metal ions such as for example, ⁶⁷Ga,¹¹In, can be non-covalently linked to epitope tag in the form of a mixedchelate with IDA, NTA, and the like. Methods for detecting or aradioactive label are well known in the art.

Magnetic beads are also another preferred detectable label. A variety ofmagnetic beads compatible with cells is known in the art. See forexample PCT patent application PCT WO 90 01,295, U.S. Pat. Nos.4,101,435, 5,262,176, 4,698,302, 5,069,216, and Weissleder et al.Radiology, 175:489-493, 1990. Polymer-coated biocompatible magneticbeads with increased magnetic susceptibility and submicron size aredescribed by Kirpotin, Chan, Bunn, U.S. Pat. No. 5,411,730. The beadstypically include magnetite or superparamagnetic iron oxide and have thesize from 5 nm (superparamagnetic beads) to several micro-m. One or moreligands are attached to the beads by a non-covalent bond or by acleavable, covalent bond. The art generally recognizes that magneticbeads can be conjugated to ligands, e.g. antibodies (Weissleder et al.Radiology, 182:381-385, 1992). After incubation with the cells, thebeads which are not internalized, including surface-bound beads, aredissociated from the cells and removed, e.g. by washing. The cells thatare capable of internalizing the ligand, and thus, have internalized themagnetic beads attached thereto, can be detected by magnitometry.Alternatively, the cells are separated using magnetic field, e.g. byhigh gradient magnetic separation (Miltenyi Biotech AG). Because of thebiocompatibility of the magnetic beads, the separated,ligand-internalizing cells are viable and can be maintained alive, e.g.in cell culture, for future research or medical use.

It is appreciated that more than one kind of ligand can benon-covalently or cleavably covalently attached to the effector, e.g.reporter or nanoparticle carrying thereof, thus, the simultaneousselection and detection of cells for internalization of multiple ligandtypes in the same batch of the cells is possible.

In certain embodiments, the effector (e.g. reporter/label) isnoncovalently linked to the ligand directly, while, in otherembodiments, the effector is contained within and/or complexed with ananoparticle and the nanoparticle is non-covalently coupled to theligand. As used herein a nanoparticle is any “vehicle” capable ofcomplexing with or containing the effector and providing a non-covalentlinkage to the ligand.

A wide variety of materials are suitable as “nanoparticles” including,but not limited to porous microbeads (e.g. controlled pore glass),lipids and liposomes, various polymers, various dendrimers, and thelike. Suitable liposomes include, but are not limited to variousliposomes including, but not limited to small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Varioustechniques for forming liposomes have been described in the literature,including but not limited to, pressure extrusion, detergent dialysis,dehydration-rehydration, reverse-phase evaporation, remote loading,sonication and other methods (see, e.g, New (1990) Preparation ofliposomes. In: R.R.C. New (ed.) Liposomes: A Practical Approach. I.R.L.Press, Oxford, pp. 33-10413). Alternatively, the effector molecule(s)can simply be complexed with a lipid.

In still other embodiments, the effectors are combined with variouspolymers such as those used as drug carriers, and the like. Examples ofsuitable polymers include, but are not limited to polyvinylpyrrolidone,pyran copolymer, polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. The substances may also be coupledto biodegradable polymers useful in achieving controlled release of adrug. Suitable polymers include polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

In still other embodiments, the effector(s) are complexed with variousdendrimers. Dendrimers are three dimensional, highly ordered oligomericand/or polymeric compounds typically formed on a core molecule ordesignated initiator by reiterative reaction sequences adding theoligomers and/or polymers and providing an outer surface that ispositively changed. These dendrimers may be prepared as disclosed inPCT/US83/02052, and U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737,4,587,329, 4,631,337, 4,694,064, 4,713,975, 4,737,550, 4,871,779,4,857,599.

Typically, the dendrimer polycations comprise a core molecule upon whichpolymers are added. The polymers may be oligomers or polymers whichcomprise terminal groups typically capable of acquiring a charge.Suitable core molecules comprise at least two reactive residues whichcan be utilized for the binding of the core molecule to the oligomersand/or polymers. Examples of the reactive residues are hydroxyl, ester,amino, imino, imido, halide, carboxyl, carboxyhalide maleimide,dithiopyridyl, and sulfhydryl, among others. Preferred core moleculesare ammonia, tris-(2-aminoethyl)amine, lysine, ornithine,pentaerythritol and ethylenediamine, among others. Combinations of theseresidues are also suitable as are other reactive residues.

Non-Covalently Coupling the Ligand to the Effector/Nanoparticle.

In preferred embodiments, the ligand is non-covalently coupled to theeffector and/or to the nanoparticle comprising the effector. Suchnon-covalent coupling can be by means of ionic interactions,coordination bonds, such as chelation bond, and/or hydrogen bonding,and/or hydrophobic interactions, and the like. In particularly preferredembodiments, the non-covalent coupling is by means of an epitope tag.

An epitope tag, as used herein refers to a molecule or domain of amolecule that is specifically recognized by an antibody or other bindingpartner. Thus, for example, in addition to epitopes recognized inepitope/antibody interactions, epitope tags also comprise “epitopes”recognized by other binding molecules (e.g. ligands bound by receptors),ligands bound by other ligands to form heterodimers or homodimers,oligo-histidine sequence having from 2 to 8 histidine residues, such asHis₆ bound by Ni-NTA, and the like.

Epitope tags are well known to those of skill in the art. Moreover,antibodies specific to a wide variety of epitope tags are commerciallyavailable. These include but are not limited to antibodies against theDYKDDDDK (SEQ ID NO: 1) epitope, c-myc antibodies (available from Sigma,St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSVepitope, the His₄, His₅, and His₆ epitopes that are recognized by theHis epitope specific antibodies (see, e.g., Qiagen), and the like. Inaddition, vectors for epitope tagging proteins are commerciallyavailable. Thus, for example, the pCMV-Tag1 vector is an epitope taggingvector designed for gene expression in mammalian cells. A target geneinserted into the pCMV-Tag1 vector can be tagged with the FLAG® epitope(N-terminal, C-terminal or internal tagging), the c-myc epitope(C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal)epitopes.

In particularly preferred embodiments, the ligand is tagged with ahexahistidine (His₆) epitope tag which is bound by a Cu, Ni, Zn, or Cocomplex of a chelator group. Preferred chelator groups includeiminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) derivatives.One particularly preferred complex for binding His₆ tags is Ni-NTA whichis readily coupled to an effector and/or to a nanoparticle comprising aneffector (see, e.g., Example 1).

One important instance of an effector/nanoparticle is a liposome. Aliposome may contain several hundreds or thousands of molecules of aneffector (e.g. reporter) which leads to increased sensitivity of themethods taught by the present invention. Methods of making liposomes andloading them with various substances, such as effectors and, inparticular, reporters, are known to those skilled in the art anddescribed in comprehensive sources (see, e.g., Liposome Technology/Ed.by G. Gregoriadis, vol. I-III, CRC Press, Boca Raton, Fla., 1993; LasicD. (1993). Liposomes: From physics to applications. Elsevier, Amsterdam,575 pp). Liposomes with attached ligands are known to bind and/or to beinternalized by certain cells (Park, et al. (1997) Adv. Pharmacology,40:399-435). To form an effector/ligand construct of the presentinvention, one can use liposomes containing, for example, NTA- andIDA-conjugated lipids.

Metal chelating lipid conjugates capable of being incorporated into alipid vesicle are generally described by Wagner, et al., U.S. Pat. No.4,707,453. Unlike high-stability metal chelates, the present invention,in preferred embodiments, uses chelating lipid conjugates that producemetal complexes of moderate (or low) stability, and typically havingfewer coordination sites than the metal, e.g. as in NTA- or IDA groups,so that the complex coordination sphere of the metal ion in the complexis incomplete, affording formation of the metal chelation bond betweenthe metal and the epitope tag of the ligand. Such chelation bonds can bereadily dissociated by the action of a commonly used, cell-sparingchelator/metal-binding agent with higher metal-binding strength, such asethylenediamine tetraacetate (EDTA) of tiethylenetriamine pentaatcetate(DTPA).

In preferred embodiments, the lipids are so conjugated as to allowformation of the metal chelation bond between hexahistidine epitope andthe NTA or IDA or other chelating group. Typically, these conjugates areprepared using an intermediate, N-(5-amino-1-carboxyalkyl)-iminodiaceticacid (see, e.g., U.S. Pat. No. 5,047,513). Examples of such NTA lipidsand IDA lipids, without limitation, are:N-(5-(1,2-dioleoyl-sn-glycero-3-succinylamido)-1-carboxypentyl)iminodiaceticacid (DOGS-NTA) (Avanti Polar Lipids, Inc., Alabama, USA),1-(N,N-dicarboxymethylamino)-3,6-dioxaoctyl-2,3-distearylglyceryl ether(IDA-TRIG-DSGE) (Northern Lipids, Inc., Vancouver, Canada),1,2-di-O-hexadecyl-sn-glycero-3-(1′-(2′-(R)-hydroxy-3′-N-(5-amino-1-carboxypentyl)-iminodiaceticacid (DHGN) (Barklis et al., EMBO J., 16:1199-1213, 1997),N^(α),N^(α)-bis(carboxymethyl)-N^(ε)-((1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamido)-succinyl)-L-lysine(NTA-DPPE), and N^(α),N^(α)-bis(carboxymethyl)-N^(ε)-(N,N-dioctadecylamido)succinyl-L-lysine(NTA-DODA) ((Schmitt et al., J. Amer. Chem. Soc., 116:8485-8491, 1994).Lipids comprising sterols, such as cholesterol linked to a metalchelation group capable of forming a chelation bond with an epitope tag,e.g. His-tag, are provided by the present invention.

Particularly preferred lipids for making liposomeeffectors/nanoparticles are metal-chelating lipids comprising ahydrophobic lipid portion, a hydrophilic polymer linked to said lipidportion, and a chelation group linked to said hydrophilic polymer. Thehydrophobic lipid portion merges within a liposome lipid layer and actsas an “anchor” capable of keeping the metal-chelating lipid linked tothe liposome during contact with a cell. Examples of such hydrophobicportion are those of the type generally employed to produce liposomes:phospholipids, such as phosphatidylethanolamine, steroids, such ascholesterol, glycolipids, sphingolipids, lind chain mono- anddialkylamines, a long chain dialkylcarboxylic acid or ester, an ester ofa polyhydroxyalcohol, such as glycerol, and the like. The chelationgroup is preferably a group that binds to an epitope tag such ashexahistidine tag. Examples of such groups are nitrilotriacetic acid,iminodiacetic acid, and their C-substituted derivatives, in the form ofthe complexes with transition metal ions, such as divalent ions of Ni,Co, Cu, and Zn. A hydrophilic polymer is typically polyvinylpyrrolidone,polyvinyl alcohol, polyvinylmethylether, polyoxazoline or substitutedderivative thereof, polyacrylic acid, amide, N-substituted amide, orester thereof, polymetacrylic acid, N-substituted amide amide, or esterthereof, hydroxyalkylcellulose, poly(oxyalkylene), polyglycerol,polyglycolic acid, polylactic acid, water-soluble polysaccharide,poly(anhydroglucose), polyaspartamide, or hydrophilic peptide sequence.Lipid-hydrophilic polymer conjugates are generally known as liposomecomponents (see, e.g., U.S. Pat. Nos. 5,631,018, 5,395,619, 5,013, 556,4,534,899).

The polymer typically has molecular weight from about 400 to about2,000,000 Dalton. The molecular weight range of the suitable polymerdepends on the molecular weight of a monomeric unit that composes thepolymer, so that the polymer would contain more than three, preferablymore than four, and most preferably at least six monomeric units.Without being bound by a particular theory, we assume that for a freemotion of the polymer chain, advantageous for the unhindered access ofthe chelation group for the epitope tag, the chain length of the polymerwould typically be equal or exceeding the length of a kinematic segmentwhich for a flexible hydrophilic polymer typically includes 4-6monomeric units or more. The metal chelation group is preferably a groupthat binds to an epitope tag such as hexahistidine tag. Examples of suchgroups are nitrilotriacetic acid, iminodiacetic acid, and theirC-substituted derivatives, in the form of the complexes with transitionmetal ions, such as divalent ions of Ni, Co, Cu, and Zn. To prepare suchlipid-polymer-chelation group conjugates, one may start with alipid-polymer wherein one or more links that forms the polymer chainbear reactive chemical groups, such as, for example, carboxylic acid,carboxylic acid active ester, e.g. N-hydroxysuccinimide ester, mixedanhydride, isothiocyanate, amine, thiol, haloid alkyl,alpha-haloidalkanoyl, cyanuric chloride, N-maleimidyl, carbonyl,hyrdazido, azido, or hydroxylamino group. Such reactive groups are knownto those skilled in the art (see, e.g., Hermanson (1996). BioconjugateTechniques. Academic Press, New York, 785 pp.). In preferredembodiments, the chelation group is be attached using a nitrilotriaceticgroup having a functionalized alkyl substitute at one of the methylenegroups (see, e.g., U.S. Pat. No. 5,047,513). The functionalizedsubstitute is typically (C₂-C₆)-alkyl, having a functional group thatreacts with the reactive group of the polymer, such as amino, thiol, orhydroxy group. Methods of making NTA-functionalized polymers aredisclosed e.g. by Seed, et. al., PCT Appl. PCT/US97/18104, WO 98/15293.Iminodiacetic acid group is attached, e.g., by conjugation of one of thecarboxyl groups of a nitrilotriacetic acid to an amine or hydroxyl groupin the polymer, or by the reaction between an amino group of the polymerand bromoacetic acid in an alkaline medium. In one embodiment theinvention provides for the poly(ethylene glycol)-lipid conjugatescontaining a terminally attached metal chelation group capable offorming a chelation bond with an epitope tag. Poly(ethylene glycol) withmolecular weight between about 300 and about 50,000, preferably betweenabout 500 and about 20,000, most preferably between about 1,000 andabout 5,000, is suitable.

Lipids commonly used to form liposomes, such as, di(C₁₀-C₂₂) alkyl-(oralkenyl-) phosphatidylethanolamines, di(C₁₀-C₂₂) alkyl-( oralkenyl-)phosphatidic acids, di(C₁₀-C₂₂) alkyl-( oralkenyl-)phosphatidyl glycerols, di(C₁₀-C₂₂) alkyl-, alkenyl-, alkanoyl,or alkenoyl, glycerols, sphingolipids, glycophospholipids, sterols,their derivatives, as well as synthetic lipid “anchors” such asdi(C₁₀-C₂₂) alkyl-( or alkenyl-)amines or similar alkanoyl amides aresuitable. Lipid-polymer-chelator conjugates are incorporated into lipidmatrix of the liposome either before, or after the liposome formation(by co-incubation with pre-formed liposomes), in the amount of 0.1-50mol % of the liposome-lipid, preferably 0.5-10 mol. %, and mostpreferably at 0.5-5 mol. % of the liposome lipid. In a particularembodiment, poly(ethylene glycol)-lipid is poly(ethyleneglycol)-conjugated DSPE, and a chelation group is NTA.

Equipping the effector liposome with an epitope-binding, e.g. metalchelation, group in the form of a lipid-polymer-epitope binding groupconjugate provides several novel and advantageous features. Thepolymer-attached epitope-binding group, being situated well away fromthe liposome surface, and due to the polymer chain flexibility, hasbetter access to the epitope tag within a macromolecule, e.g. arecombinant protein, thereby improving sensitivity of the methods ofthis invention. Significantly, the lipid-polymer-NTA-Ni conjugate, beingmicellarly soluble in aqueous medium, could be “captured” intopre-formed liposomes pre-loaded with the effector, e.g. cytotoxin orreporter, by mere co-incubation of the liposomes and the conjugate in anaqueous buffer.

It was a surprising discovery of this invention that after co-incubationwith the effector liposomes, the epitope (e.g. hexahistidine)tag-binding activity of the liposome-captured lipid-polymer-NTA-Ni²⁺conjugate was surprisingly well preserved, as evidenced by the selectiveinternalization of cytotoxin carried by such liposomes into theHER2-receptor-bearing cells in the presence of an anti-HER2 scFvantibody having a hexahistidine epitope tag (see Example 4 below). Theliposome having an epitope-binding group attached away from its surface,e.g. via a polymer spacer, afforded co-inclusion into the liposome of apolymer-derivatized lipid which reduces liposome aggregation, reducesbackground (non-specific) binding of the liposomes to cells, and whenapplied into the body, increases the liposome longevity in circulation(U.S. Pat. No. 5,013,556), providing for better binding of theeffector/ligand constructs to the “test” cells in vivo. Typically, forreducing aggregation, the amounts of the lipid-hydrophilic polymerconjugate of 0.1-0.9 mol. % of total lipid are sufficient, while forincreasing circulation longevity, the concentrations of 1-20 mol. % oftotal lipid are needed (U.S. Pat. No. 5, 013,556). The use of epitopebinding group, e.g. NTA, attached to the liposome via a polymer spacer,unexpectedly resulted in a remarkably better loading of the liposomewith an effector, such as cytotoxin doxorubicin, into the effectorliposome (see Example 4).

In certain embodiments, the effector can be covalently coupled to theligand providing such coupling is readily and/or specifically cleaved.Preferably the cleavage occurs under the conditions which preserve thestructural integrity of a cell such as not to allow the internalizedeffector, e.g. reporter, to leave the cell in the course of, or as aresult of, the cleavage. Such cleavable couplings are well known tothose of skill in the art. For example, in one embodiment, a linkercomprising a nucleic acid restriction site or a protease recognitionsite is readily cleaved by application of the appropriate endonucleaseor protease.

Other cleavable linkers are well known to those of skill in the art(see, e.g., U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014). Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. Examples of cleavable linkers include, without limitation,enamine, hydrazone, oxazolidine, ketal, acetal, ortho-esters of carbonicacid, thioesters, substituted hemiesters and hemiamides of 2-alkyl or2,3-dialkyl substituted maleic acid, and vinyl ethers that dissociate byhydrolysis in physiological aqueous solutions at pH 3-6; disulfide bondsthat dissociate in the presence of thiolytic agents (cysteine,mercaptoethanol, dithiotheitol, tris-carboxymethylphosphine, and thelike, typically at 0.1-10 mM concentration) in aqueous solutions underphysiological conditions of pH and salinity; ester bonds that arecleaved by base-catalyzed or enzymatic hydrolysis in aqueousphysiological buffers at pH 7-9, and the bonds that are cleaved byphotodissociation, such as 2-nitrobenzyl derivatives (Haugland (1996).Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes,Inc., Eugene, Oreg. 6th Ed.).

Contacting a Cell with the Ligand/Effector.

In preferred embodiments, one or more “test cells” (e.g., cells that areto be screened for the ability to internalize a ligand) are contactedwith the effector (e.g. reporter) non-covalently coupled to a ligand.Often a cell will be contacted with a number of differentligand/effector combinations. Contacting is typically under conditionsin which the cell is capable of internalizing a ligand; that is, forexample, under condition where an internalizing receptor is capable offunctioning. In the case of mammalian cells, incubation at the slightlyelevated temperature (30-40° C.) in an aqueous solution withphysiological balanced salts and cell nutrients is preferred. Typicallythe cell is contacted (e.g. incubated) with the ligand/effectorconstruct in culture although such contacting can be with cells derivedfrom an acute/fresh preparation.

While such contacting is typically accomplished ex vivo it is recognizedthat, in certain embodiments, the contacting can be accomplished invivo. It is noted that U.S. Pat. No. 6,068,829 discloses methods ofidentifying molecules that home to a selected organ in vivo. The methodsinvolve transfecting a living organism with a library of ligands andidentifying the ligands that localize to a particular tissue. Thispatent thus demonstrates the feasibility of contacting a cell in vivowith a heterologous ligand.

In particularly preferred embodiments, the ligand and the effector arejoined through an epitope tag. In such embodiments, formation of thenon-covalent linkage (between ligand and effector) and contacting of thecell with the ligand/effector construct can be easily combined into asingle procedure. By way of illustration, Example 1 describes theincubation of NTA-liposomes, i.e. liposomes having surface-attachedNi-NTA groups, (0.5-1 mM total phospholipid) were incubated for 4 hourswith the cells along with a (His) ₆-containing ligand (˜20 micro-g/mL)in 100 micro-L tissue culture media supplemented with 10% FCS at 37° C.Under such conditions, the ligands formed non-covalent linkages with theeffector and were internalized by the cells.

Virtually any cell can be used with the methods of this invention. Suchcells include both eukaryotic and prokaryotic cells. Bacterial cells,fungal cells, algal cells, plant cells, animal cells are all well suitedto the methods of this invention. In particularly preferred embodiments,the cells are vertebrate cells, more preferably mammalian cells, andmost preferably human cells. The cells can be cultured ex vivo, obtainedfrom fresh preparations, present in a tissue culture, or in a tissue invivo. In high-throughput screening applications, cultured cells are mostpreferred.

Dissociating the Effector from the Ligand.

In preferred methods, after the ligand/effector construct has beencontacted to the cell for a time sufficient to allow ligandinternalization, the effector is separated from the ligand bydissociating the non-covalent attachment. This is accomplished by any ofa number of methods well known to those of skill in the art. Methods ofdisrupting such non-covalent attachments include but are not limited tothe use of dissociating factors and/or agents, such as heat, acidity,chaotropic agents, high salt, chelating agents, and the like.Particularly preferred are cell-sparing methods where the integrity ofthe cell is preserved so that after the dissociation the internalizedligand and/or effector remain essentially within the cells. If aneffector and a ligand are linked by a metal chelation bond such asbetween Ni-NTA group and a His₆-epitope tag, the dissociating agent ispreferably a reagent that binds a divalent transition metal ions, forexample, strong chelator such as EDTA typically at low concentration of0.2-5 mM, a weak metal complex-forming agent, such as imidazole, at highconcentration, typically 100-300 mM, or a dithiol compound such as2,3-dimercaptosuccinate, typically at 0.2-10 mM in a neutralphysiological saline buffer. By competing for binding of a metal ion,e.g. Ni2+, with the ligand-liposome chelation bond, these dissociatingagents deprive the bond of the metal ion causing the bond to break down.In one preferred embodiment, as illustrated in Example 1, cellsurface-attached liposome/ligand complexes were dissociated, and thedissociated liposomes were removed by washing the cells 3-4 times with adissociating buffer, in this case phosphate-buffered physiologicalsaline containing 2 mM MgCl₂, 2 mM CaCl₂, and 1 mM EDTA or with 250 mMphosphate buffered imidazole (pH 7.4). Because the internalizedliposome/ligand complexes were inaccessible to the dissociating buffer,the internalized liposomes remained in the cells providing thedetectable signal that indicated the presence and the amount of theinternalized ligand/liposome construct.

Dissociating can involve releasing an effector, e.g. a reporter, fromthe nanoparticle by disrupting it, so that the released effector can bewashed away from the cells. If the nanoparticle is a liposome, thereleasing buffer can include a liposome-destabilizing factor. Liposomeswith triggered release of encapsulated agents, induced by chemical ofphysical factors, for example, by briefly subjecting to pH 4-6,thiolytic agents, mild heating (42-45° C.), or light, are known in theart (see, e.g., Gerasimov et al. (1995) Vesicles, Ch. 17, p.679-746;Kirpotin, et al. (1996), FEBS Lett., 388:115-118). When the effector isdissociated from the ligand by disrupting a nanoparticle, e.g. aliposome, the ligand may be in the form of an amphipathic conjugate,such as lipid- or lipid-hydrophilic polymer conjugate, in which case anon-covalent bond between the ligand and the effector is by hydrophobicinteractions.

Cleavable, covalent attachments between the ligand and the effector canbe also destroyed in a cell-sparing matter. For example,carbonylhydrazone bonds are formed between a carboxy acid hydrazidegroup attached to a reporter, e.g., liposome, or a magneticnanoparticle, and a ketone or aldehyde group produced by the periodateoxidation of a N-terminal serine or treonine engineered into arecombinant protein/peptide ligand. In the acidic aqueous environment(pH 3-6), the bond is hydrolyzed to release the reporter.

Detecting the Internalized Ligand.

The internalized ligand is detected according to methods well know tothose of skill in the art. The ligand can be detected directly (e.g.through various purification techniques), however, in a preferredembodiment, the ligand is detected by detecting the effector moleculeattached to (or associated with) the ligand. Where the effector is areporter (detectable label), the effector is detected using methodstypically used to detect a label of the same kind. Thus, where theeffector is a radionuclide, detection is by methods such asscintillography, or autoradiography. Where the effector is acolorimetric tag, detection is by optical means. Where the effector is afluorescent tag, detection is by methods such as fluorimetry, flowcytometry, or fluorescent microscopy. When the effector is a magneticbead, detection is by magnetometry.

Where the effector is a cytotoxin, detection of internalization caninvolve a measurement of cell mortality. Conversely, where the effectoris a growth factor or a mitogen, detection can involve detection of cellgrowth or proliferation.

Assays for ligand internalization are typically scored as positive wherethere is a detectable signal from an internalized effector, preferablyas compared to a negative control. In a preferred embodiment, to score apositive result the difference between the internalized “test” assay andthe (usually negative) control is statistically significant (e.g. atgreater than 80%, preferably greater than about 90%, more preferablygreater than about 98%, and most preferably greater than about 99%confidence level), e.g, as determined using any statistical test suitedfor the data set provided (e.g. t-test, analysis of variance (ANOVA),semiparametric techniques, non-parametric techniques (e.g. WilcoxonMann-Whitney Test, Wilcoxon Signed Ranks Test, Sign Test, Kruskal-WallisTest, etc.). Most preferred “positive” assays show at least a 1.2 fold,preferably at least a 1.5 fold, more preferably at least a 2 fold, andmost preferably at least a 4 fold or even a 10-fold difference from thenegative control.

Detecting can include quantitative determination (quantification) of theinternalized ligand so that more precise comparison between variousligands as to their internalizing capacity can be made. Methods forquantification of the effector molecules such as cytofectins, enzymes,fluorescent, light-absorbing, radioactive, or magnetically susceptiblesubstances, are known in the art (see e.g. Spector, et al. Cells. ALaboratory Manual, vol. 1-3, Cold Spring Harbor Laboratory Press, 1998).

Identifying the Internalizing Receptor(s).

The assays described above, can also be used to identify (e.g.previously unknown) internalizing receptors. In preferred embodiments,such methods involve identifying internalized ligands according to themethods described above. The internalized ligands are recovered from thecell and/or identified. The recovered and/or identified ligand can thenbe used to identify the receptor that internalized that ligand.

Methods or recovering internalized ligands are well known to those ofskill in the art. This can involve lysing the cell and performingstandard purification methods to isolate the labeled (effector-bound)ligand. Methods of purifying molecules from cells are well known tothose of skill in the art. Typical purification methods include, but arenot limited to gel electrophoresis, anion exchange chromatography (e.g.Mono-Q column, Pharmacia-LKB, Piscataway, N.J., USA), or reverse phasehigh performance liquid chromatography (HPLC). For a review of standardtechniques see, Methods in Enzymology Volume 182: Guide to ProteinPurification, M. Deutscher, ed. (1990), pages 619-626.

Alternatively, after the cell is lysed, the ligand can be dissociatedfrom the effector and the epitope tag on the ligand can then be used torecover the ligand by affinity chromatography. Thus, for example, wherethe ligand is affinity tagged with a His₆ tag, the ligand can berecovered e.g., with an Ni-NTA affinity column, Ni-NTA gel, or Ni-NTAconjugated magnetic beads (see, e.g., QIAexpress™ Detection and AssayHandbook, Qiagen).

Detecting the Cells that Internalize a Ligand.

When the method of the invention is used to detect the cells thatinternalize a ligand, following the step of dissociating the effector,e.g. a reporter or nanoparticle, from the ligand, the presence of theeffector in the cells is detected by any means known to those skilled inthe art (see, e.g., “Detecting the Internalized Ligand” above). Incertain preferred embodiments, the detection methods involve examinationof individual cells. Examples of such methods include, in the case of afluorescent reporter, flow cytometry and fluorescent microscopy, and thelike; in the case of a radionuclide reporter, autoradiography, and thelike.

Detection of the ligand internalized in the cells can involve isolatingthe ligand-internalizing cells from those that do not detectablyinternalize the ligand. Following the dissociation of the effector, e.g.a reporter or nanoparticle, from the ligand, the ligand-internalizingcells can be isolated, for example, in the case of fluorescent reporter,by fluorescence-activated cell sorting (FACS), or in the case of thereporter being a magnetic bead, by high gradient magnetic separation.The isolated cells are then examined or utilized e.g., for research,industrial, or medical purposes.

One particularly preferred embodiment of this method involves detectionof malignant cells in the body tissue or fluid samples from a patient.In this case, ligands that are selectively internalized by malignantcells, are used. For example, antibodies, such as scFv, can be selectedfor specific internalization into malignant or other pathological cellsas described herein and in the co-pending, co-owned U.S. patentapplication Ser. No. 09/249,529, and used according to the presentinvention to detect and/or select the pathological cells in the samplesof a patient body tissues or fluids.

Detecting Binding and Internalization of a Ligand by the Cells.

The methods of this invention also can be used for detecting bothsurface-binding and internalization of a ligand by a cell. In preferredembodiments, the methods can include contacting a cell with theligand/effector (e.g. ligand/reporter) construct, removing a portion ofthe construct which is not associated with the cell, i.e. that isneither surface-bound, nor internalized, by the cells, and detecting thereporter associated with the cell to obtain a first reading indicating atotal amount of the ligand which is bound to the cell surface andinternalized by the cell. The removal of the non-cell associated portionof the reporter is preferably by removal of the ligand/reporterconstruct achieved by washing the cell under non-dissociatingconditions, such as using phosphate-buffered saline, buffered balancedsalt solution (Ringer, Hanks), cell growth medium, or otherphysiological medium without a dissociating agent. Thus, in the absenceof these agents, the cell surface-bound ligand/effector constructs willremain intact on the cell and contribute to the signal detected from theeffector providing the first measurement of the total cell-associatedligand. Then the reporter is dissociated from the ligand in asurface-bound ligand/effector constructs, and dissociated effector isremoved from the surface of the cell, for example, as in the case ofNTA-Ni-His₆-linked ligand/effector, by washing the cell with aphysiological buffer containing a divalent metal ion-binding agent asdescribed herein. Then the reporter remaining in the cell is detectedproviding a second measurement of an amount of the ligand/effectorconstruct that is internalized. The difference between the firstmeasurement and the second measurement corresponds to the amount of theligand bound to cell surface but not internalized. In some cases, beforetaking the first measurement it is advantageous to arrest theinternalization process without disintegrating the cell. This is readilyachieved by treatment of the cell with a metabolic inhibitor, such asanhydroglucose or sodium azide, or by decreasing the temperature(chilling on ice) typically to less than 10° C., typically to about 0-4°C.

Screening for Modulators of Internalization.

The methods of this invention can also be used to screen for agents thatmodulate the internalization of a ligand or ligands. In preferredembodiments, these methods entail screening for ligand internalizationas described herein where the cells are contacted before, and/or during,and/or after the time they are contacted with the effector/ligandconstruct with the test agent(s) to be screened. A difference in ligandinternalization by cells contacted with the test agent(s), e.g. ascompared to negative controls comprising the test agent(s) at a lowerconcentration or the absence of the test agent(s), indicates that thetest agent(s) modulate (e.g. increase or decrease) internalization thesubject ligand(s). An increase of internalized ligand indicates that thetest agent(s) upregulate internalization, while a decrease ininternalized ligand indicates that the test agent(s) downregulateinternalization.

Depending on the duration of the assay, the increase or decrease canrepresent an increase or decrease in total ligand internalized or inrate of internalization (i.e. amount of ligand internalized per unittime). In still other embodiments, the ligand(s) can be screened for theability to alter the time-course of internalization.

The assays for modulator activity are typically scored as positive wherethere is a difference between the activity (signal) seen with the testagent present and the (usually negative) control, preferably where thedifference is statistically significant (e.g. at greater than 80%,preferably greater than about 90%, more preferably greater than about98%, and most preferably greater than about 99% confidence level). Mostpreferred “positive” assays show at least a 1.2 fold, preferably atleast a 1.5 fold, more preferably at least a 2 fold, and most preferablyat least a 4 fold or even a 10-fold difference from the negative control(experiment where the test agent is absent or present at a lowerconcentration).

High Throughput Screening.

The methods of this invention are well suited for high throughputscreening. Particularly where an epitope tag is used to link the ligandto the effector, the assays can essentially be run in a “single step”format without elaborate purification of the ligand and/or the effector.As shown in example 1 it is sufficient to combine the subject cell(s)with the ligand and the effector at once under appropriate “incubation”conditions. The ligand joins to the effector and if the cell has acorresponding internalizing receptor the ligand is internalized into thecell along with the bound effector (e.g. label).

The cells utilized in the methods of this invention need not becontacted with a ligand and/or single test agent at a time. To thecontrary, to facilitate high-throughput screening, a single cell may becontacted by at least two, preferably by at least 5, more preferably byat least 10, and most preferably by at least 20, at least 50 or even atleast 100 ligands or test compounds. If the cell scores positive, it canbe subsequently tested with a subset of the ligands or test agents untiltest agents having the activity or the internalized ligands areidentified.

High throughput assays for various reporter gene products are well knownto those of skill in the art. For example, multi-well fluorimeters arecommercially available (e.g., from Perkin-Elmer). Other high throughputscreening systems are commercially available (see, e.g., Zymark Corp.,Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols the various high throughput. Thus, for example, Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

High throughput screens can be performed in a number of formats known tothose of skill in the art. In preferred embodiments, high throughputscreens utilize a microtiter plate format (e.g. a 96 well format, a 480well format , a 960 well format, etc.).

Delivery of Effectors into Cells.

In certain embodiments this invention provides a composition fordelivery of an effector into a cell, which composition comprises (i) ametal-chelating lipid comprising a hydrophobic lipid portion, ahydrophilic polymer linked to said lipid portion, and a chelation grouplinked to said hydrophilic polymer wherein the chelation group iscomplexed to a metal ion and binds to an epitope tag, and (ii) a ligandcomprising said epitope tag, where the epitope tag comprises a sequenceof at least two neighboring histidine residues (a histidine tag), andwhere effector is associated with said metal-chelating lipid. The tagpreferably comprises six neighboring histidine residues (hexahistidinetag). A preferred composition is one where the metal-chelating lipid andthe effector are comprised in a liposome. Any effectors and/or ligandsdescribed herein are suitable. The effector is for example, a reporter,a cytotoxin, a drug, or a nucleic acid. The ligand is typically aprotein, a carbohydrate, a nucleic acid, of a small organic molecule.The ligand may be natural or synthetic. Preferred protein ligands arethose that comprise the antigen-binding sequences of an antibody, suchas immunoglobulins and fragments thereof, both naturally andrecombinantly produced, including single-chain fragments. The liposomemay further comprise a lipid-polymer conjugate, particularly, alipid-poly(ethylene glycol) conjugate. In the liposome, themetal-chelating lipid typically constitutes between 0.1 mol % and 50mol. % , preferably between 0.2 mol. % and 10 mol %. Optionally, thelipid-polymer conjugate (without the metal-chelating group) can beincluded to up to 20 mol % of the liposome lipid.

Databases of Internalizing Ligands and/or Internalizing Receptors.

In certain embodiments, the methods of this invention further compriselisting the identified internalizing receptors in a database identifyinginternalizing receptors and/or listing modulators of ligandinternalization in such a database. The term database refers to a meansfor recording and retrieving information. In preferred embodiments thedatabase also provides means for sorting and/or searching the storedinformation. The database can comprise any convenient media including,but not limited to, paper systems, card systems, mechanical systems,electronic systems, optical systems, magnetic systems or combinationsthereof. Preferred databases include electronic (e.g. computer-based)databases. Computer systems for use in storage and manipulation ofdatabases are well known to those of skill in the art and include, butare not limited to “personal computer systems”, mainframe systems,distributed nodes on an inter- or intra-net, data or databases stored inspecialized hardware (e.g. in microchips), and the like.

Kits.

In another embodiment, this invention provides kits comprising materialsfor the practice of the methods described herein. In one preferredembodiments the kits comprise a container containing a ligandnon-covalently coupled to a effector (e.g. a reporter) through anepitope tag. The kit may comprise a “single” construct having one typeof ligand or a library of constructs providing a multiplicity ofdifferent ligands. Alternatively, an effector, e.g. a reporter ornanoparticle, can be provided, along with one or more ligands, inseparate containers, so that the effector/ligand construct will beformed when the effector and the ligand are combined by a user accordingto the provided instructions and the needs of a particular application.

The kit can optionally include other instruments and/or reagents forpractice of the methods of this invention. Such reagents and instrumentsinclude, but are not limited to microtiter plates, cells, buffers,filters for detection of fluorescent labels, software for running assayson high throughput robotic systems, and the like.

In addition, the kits can include instructional materials providinggeneral directions and/or specific protocols for the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media caninclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 A Novel Assay for Monitoring Internalization of AntibodiesUsing Metal-Chelating Liposomes

Introduction

Antibodies and antibody fragments can deliver a variety of agents suchas drugs, genes, toxins or radionuclides to target cells expressing theantigen. Endocytosis of the antibody fragment to the interior of thecell can in many cases increase the therapeutic effect of thetherapeutic agent. A major advantage of receptor mediated endocytosis asa drug delivery route is that therapeutic agents can be deliveredspecifically into target cells that overexpress the receptor and therebyincrease efficacy while reducing systemic toxicity. For example,anti-ErbB2 antibodies have been used to target doxorubicin containingliposomes (Park et al. (1995) Proc. Natl. Acad. Sci., USA, 92:1327-1331) or Pseudomonas exotoxin (immunotoxin) into the interior oftumor cells (King et al. (1996) Semin Cancer Biol 7: 79-86).

The majority of antibodies generated by immunization do not bind toreceptors in a manner that triggers endocytosis (Hurwitz et al. (1995)Proc. Natl. Acad. Sci., USA, 92: 3353-3357). Thus it is desirable to beable to screen for antibodies that can elicit the desired response. Themost common method for monitoring internalization of ligands andantibodies into cells involves radiolabeling of the protein and employsa low pH buffer (usually glycine-HCl pH 2.8) in order to dissociatesurface bound antibody. However, reports from several laboratoriesindicate that this buffer in some cases only partially dissociatesantigen-antibody complexes and therefore can introduce majorinaccuracies in internalization experiments (Matzku et al. (1990) Br. J.Cancer Suppl 10: 1-5; Tsaltas and Ford (1993) Immunol Invest, 22: 1-12).Alternatively, antibodies can be biotinylated with NHS-SS-biotin andincubated with live cells. Following specific reduction of biotin groupson cell surface bound antibody with reducing agent, internalization maybe quantified by immunoblotting (Liu et al. (1998) Cancer Res 58:4055-4060). However, the accuracy of this method also relies on completeremoval of biotin from the cell surface bound antibody. Another drawbackof these methods is that they rely on laborious labeling of each ligandprotein allowing only a limited number of different antibodies to bescreened for internalization. The direct labeling of the protein oftenalso results in loss of binding activity of the antibody or ligand. Inaddition, the stringent conditions that are required to strip the cellsurface in these procedures may affect cell viability.

In this example we report a novel assay for internalization termed“Chelated Ligand Internalization Assay” (CLIA). Liposomes wereformulated with Ni²⁺-NTA-lipids capable of binding (His)₆-taggedproteins. The NTA containing liposomes were loaded with fluorescent dyeand mixed with a number of different (His)₆ containing anti-receptorantibody fragments or intact antibody complexed to (His)₆-tagged ProteinA. For those antibodies that bind weakly to protein A, protein G can beused instead. Internalization of the scFv/liposome/receptor complex wasdetected by fluorescence microscopy or fluorimetry after gentle removalof the liposomes from cell surface bound complexes using EDTA. Cellularuptake of the complex was dependent on the specificity of the scFv aswell as the ability of the antibody fragment to trigger internalizationrequiring <50,000 receptors/cell for detection. The assay requires onlyminute amounts of antibody fragment and was also performed using crude,unpurified supernatants of E coli expressing the antibody fragment.

Methods

Liposome Preparation

Liposomes are prepared from 1-palmitoyl-2-oleoyl-phosphatidylcholine(POPC) and cholesterol (6:4 molar ratio) and varying amounts of NTA-DOGS(Avanti Lipids; 0.5-5 mol. % of POPC amount) by lipid film hydration insolution containing 35 mM 8-hydroxypyrene-1,3,5-trisulfonic acid sodiumsalt (HPTS) (Molecular Probes Inc., Oregon, USA), pH 7.0, adjusted tothe osmolality of 280 mmol/kg with NaCl. In some cases, the liposomeswere made using 1,2-distearoyl-phosphatidylcholine (DSPC) instead ofPOPC, and the lipophilic fluorescent labels DiIC1₈(3)-DS andDiIC1₈(5)-DS (0.1-1 mol. % of the liposome phospholipid) were usedinstead of HPTS, with the same results. In these cases, hydration is at55-60° C. in an aqueous 140 mM NaCl buffered with 5-20 mM4-(N-2-hydroxyethyl-piperasino)ethylsulfonic acid sodium salt (HEPES) topH 7.2-7.4. After hydration, liposomes are formed by membrane extrusionthrough two 0.1 μm polycarbonate membranes (Corning) as described(Kirpotin et al. (1997) Biochemistry 36: 66-75). Un-encapsulated HPTSwas then separated by gel-filtration on a cross-linked dextran beads(SEPHADEX G-25) (Pharmacia Amersham, New Jersey, USA) column.

ScFv Expression and Purification

The scFv's C6.5 (anti-HER2) (Schier et al. (1995) Immunotechnology, 1:73-81), and F5 (anti-HER2) (PCT/US99/07395) were cloned into expressionvector pUC119mycHis (Schier et al. (1995) Immunotechnology, 1: 73-81)and expressed in E. coli TG1. Briefly, 0.75 L of media (2×TY with 100μg/mL ampicillin and 0.1% glucose) was inoculated 1/100 with anovernight culture. The culture was grown to an A₆₀₀ of 0.9 andexpression was induced by the addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of0.5 mM. The culture was then incubated at 30° C. for an additional fourhours.

Cells were harvested by centrifugation (4000×g, 20 min.) and the pelletswere resuspended in periplasmic extraction buffer (PPB) (30 mM Tris, 2mM EDTA, 20% sucrose, pH=8) containing 100 μg/mL DNase and incubated onice for 30 min. The bacteria were pelleted by centrifugation at 5000×gfor 20 min. The pellets were resuspended in osmotic shock buffer (5 mMMgSO4) and incubated for another 20 min on ice. The bacteria werepelleted (7000×g, 20 min.) and supernatants from the PBB and MgSO₄fractions were combined and cleared by centrifugation at 10000 rpm for30 min at 4° C. The resulting solution was dialyzed in PBS (two changes,4 L PBS pH 8). All molecules were purified by immobilized metal affinitychromatography (IMAC) (Qiagen) followed by desalting on a cross-linkeddextran gel exclusion PD10 column (Pharmacia Amersham, N.J., USA).Protein concentrations were determined spectrophotometrically from theabsorbance at 280 nm (A₂₈₀ using the absorbance value of 1.4 for 1 mg/mLprotein solution in a 1 cm cuvette.

For induction in microtitre plates, wells containing 150 μl of 2× TYcontaining 100 μg/ml ampicillin and 0.1% glucose were inoculated with anovernight culture of E coli TG1 with the plasmid containing the scFv.Cultures were grown to an A₆₀₀˜1, and scFv expression induced by theaddition of IPTG to a final concentration of 1 mM. Bacteria were grownovernight at 30° C., the cells removed by centrifugation, and 30 μL ofthe supernatant containing scFv used directly in the internalizationassay.

Preparation of Protein A-(His)₆ Conjugate

Protein A was conjugated to the (His)₆-containing peptide CGGGHHHHH (SEQID NO:2) using the bifunctional reagentm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS; Pierce).2 mg of Protein A was treated with 0.2 mg Sulfo-MBS in PBS for one hourat room temperature. Unreacted Sulfo-MBS was removed by gel filtrationand the protein then reacted with 0.2 mg of the (His)₆-containingpeptide in PBS for one hour at room temperature and unreacted peptidewas removed by gel-filtration.

Assay Procedure

Human breast cancer cells SKBR3, SKOV3, BT474, MCF7, MDA-MB-453,MDA-MB-468 (American Type Culture Collection, ATCC) were grown to 80-90%confluence in the media type recommended by ATCC supplemented with 10%fetal calf serum (FCS) and harvested by trypsinization using standardtechniques. Cells were seeded in 96-well plates at 10,000 cells/well andincubated overnight at 37° C. The next day, NTA liposomes (0-1 mM totalphospholipid) were incubated for 4 hours with the cells along with the(His)₆-containing ligand (20 μg/mL unless otherwise indicated) in 100micro-L tissue culture media supplemented with 10% FCS. Whensupernatants of induced E. coli cultures were used in the assay, 65 μLof cell culture media containing 10% serum and NTA-liposomes were mixedwith 35 μL of supernatants. To test the internalization of monoclonalantibodies, which do not contain a (His)₆-tag, 10 μg/mL of ProteinA-(His)₆ was used to complex 40 μg/mL of a recombinant, humanizedanti-HER2 monoclonal IgG HERCEPTIN (Genentech, Inc. California, USA). Tostrip cell surface of the liposomes linked within un-internalizedliposome/ligand complexes, cells were washed 3-4 times with 170 μL PBScontaining 2 mM MgCl₂, 2 mM CaCl₂, and 1 mM EDTA or with 250 mMphosphate buffered imidazole (pH 7.4). Cells were lysed in 50 μL 0.01 MNaOH before reading fluorescence at 460/530 nm in a RC4 microfluorimeter(BIOTEK).

Cell Surface Binding Measurements

Cells were harvested by trypsinization using standard techniques. The F5was incubated in triplicate with 1×10⁵ cells in 96-well plates withV-shaped wells for two hours at concentrations indicated. Cell bindingwas performed at room temperature in PBS containing 2% FCS and 0.1%sodium azide in a total volume of 200 μL. After two washes with 200 μLPBS, bound scFv was detected by the addition of 100 μL (10 μg/mL) ofFITC labeled anti-FLAG MAb M1 (Sigma). After a 30 minute incubation atroom temperature, the cells were washed twice and resuspended in PBScontaining 4% paraformaldehyde. Fluorescence was measured by flowcytometry in a FACSort (Becton-Dickinson) and median fluorescence (F)was calculated using Cellquest software (Becton-Dickinson) and thebackground fluorescence subtracted.

Results Liposome Formulation

Liposomes were formulated with 0, 0.5, 2 and 5 mol. % NTA-lipid andtested for internalization into SKBR3 tumor cells using an anti-ErbB2scFv antibody (F5) engineered to contain a C-terminal (His)₆-tag. Afterfour hours of internalization, cells were washed with 1 mM EDTA in PBS,lysed in base and the fluorescence read in a microfluorimeter. Theintensity of the signal increased dramatically with increasing NTA-lipidcomposition for the range tested (FIG. 1). The internalization wasabolished when the scFv did not contain a his-tag or when liposomes wereformulated without NTA containing lipid (FIG. 2). To expand the utilityof the assay to full-length monoclonal antibodies, Protein A wasconjugated to the peptide CGGGHHHHHH (SEQ ID NO:3) using thebi-functional reagent sulfo-MBS which cross links the thiol group in thepeptide to primary amines on Protein A. SDS-PAGE analysis confirmedsuccessful conjugation of multiple peptides per Protein A molecule asdemonstrated by an apparent shift in molecular weight of approximately10 kDa (results not shown). When SKBR3 cells were co-incubated withProtein A-(His)₆ and the monoclonal anti-ErbB2 antibody Herceptin,NTA-liposomes were specifically endocytosed (FIG. 2). Protein A-(His)₆or Herceptin alone did not increase the uptake of NTA-liposomes,indicating that it is mediated by the Herceptin/Protein A-(His)₆ complex(FIG. 2).

Assay Optimization

The effect of increasing the liposome concentration in the reaction wasinvestigated using the anti-ErbB2 scFv antibody (F5) or an irrelevantantibody binding a vascular antigen not expressed on SKBR3 cells. Thecellular uptake of liposomes was proportional to the concentration ofliposomes in the reaction. In the 0-800 μM phospholipid range tested,the non-specific antibody did not internalize liposomes above background(FIG. 4).

Sensitivity of the Assay

The sensitivity of the assay was tested with varying concentrations ofseveral antibodies to different epitopes on SKBR3 cells, FIG. 4. Onlythe F5 scFv antibody (anti-ErbB2) resulted in internalization of thecomplex. Interestingly, the irrelevant antibody (4G7) and thenon-internalizing anti-ErbB2 antibody (C6.5) did not mediateinternalization of the NTA-liposomes (FIG. 5). This s is consistent withprevious results we obtained by confocal microscopy analysis of theinternalization F5 and C6.5 The detection level of the assay with the F5scFv on SKBR3 cells was below 1 μg/mL of purified antibody.

Assay Does Not Require Antibody Purification

Because of the specific interaction of the (His)₆-tag with NTA on theliposome, the assay should permit the use of unpurified scFv, allowing alarge number of scFv molecules to be assayed for internalization. Totest this, soluble scFv expression was induced from E. coli in 96-wellculture plates and the supernatant tested for activity on live SKBR3cells using 5 mol. % NTA liposomes. Previous experiments (results notshown) had determined that SKBR3 cells tolerate as much as 50% bacterialculture supernatant for up to 24 hours. Supernatants of E coliexpressing the F5 scFv were mixed 1:3 with cell culture media containing10% serum and antibiotics as well as 500 μM NTA liposomes and incubatedwith live SKBR3 cells. Results were similar to results obtained with 20μg/mL of purified scFv with similar specificity (FIG. 6).

Profiling Tumor Cell Lines for Antibody Internalization

The scFv antibody to EGFR (C10) was used to profile a panel of breastcancer cell lines and CHO transfectants (FIG. 7). Only the cell lineMD-MDA 468 and CHO cells transfected with EGFR internalized significantamounts of NTA-liposomes. The specificity of the assay is exemplifiedwith C10 internalizing into CHO and CHO transfected with EGFR. Uptake ofthe fluorescent NTA liposomes into the EGFR transfected CHO cells was165 times that of the untransfected.

The profile of F5 internalization largely correlated with cell surfaceexpression of ErbB2 as determined by FACS with the F5 antibody (FIG. 8).However, the cell line SKOV3 did not take up as much liposome as wouldbe expected from its cell surface expression level of ErbB2. The poorinternalization of ErbB2 into this cell line has been describedpreviously (Kirpotin et al. (1997) Biochemistry 36: 66-75). When totaluptake into the same panel of cell lines was determined usingF5-liposomes (in which the antibody is covalently coupled to the lipidvia non-cleavable bond) the discrepancy with F5 scFv binding by FACS wasless pronounced. This is most likely due to cell surface boundF5-liposomes, and thus when there is a covalent, non-cleavable bondbetween the ligand and the effector, this assay does not effectivelymeasure internalization.

Example 2 Lipid-NTA Conjugates for Epitope-Mediated Non-CovalentConjugation of Ligands to Liposomes6-(1,2-Dipalmitoylglycerol-3-succinyl)amido-2-(N,N-dicarboxymethylamino)-hexanoicacid nickel salt (DPGS-NTA-Ni)

6-amino-2-(N,N-bis-carboxymethylamno)hexanoic acid (I) was synthesizedfrom N(epsilon)-CBZ-lysine and bromoacetic acid according to Schmitt etal. (1994), J. Amer. Chem. Soc. 116:8485-8491, except that removal ofcarbobenzoxy protecting group was in 4 M HBr/glacial acetic mixtureovernight, resulting in the recovery of I as a hydrobromide.

1,2-dipalmitoyl-3-succinyl-rac-glycerol (II) was prepared from1,2-dipalmitoyl-glycerol, succinic anhydride, and 4-pyrrolidinopyridineaccording to Silvius & Leventis (1987), Biochemistry, 26:3297.

DPG-NTA-Ni (III): 335 mg (0.5 mmol) of II was dissolved in 2.5 mL ofanhydrous chloroform and 1.25 ml of anhydrous dimethoxyethane. Withstirring, 66 mg (0.575 mmol) of N-hydroxysuccinimide were added,followed by the solution of 108 mg (0.525 mmol) ofdicyclohexylcarbodiimide (DCC) in 0.6 ml chloroform. After 4 hourstirring at room temperature, the precipitated urea was filtered out,and the filtrate was brought to dryness under reduced pressure. The dryresidue was suspended in the mixture of 1 ml chloroform and 3 ml ofanhydrous methanol, and 250 mg of I hydrobromide were added, followed by0.35 ml (5 mmol) of triethylamine. The mixture was brought to 50° C. toeffect dissolution of the suspended solid, and stirred at roomtemperature overnight. The mixture was diluted with 10 ml chloroform andwashed 3 times with 40 ml of the 50% aqueous methanol containing 0.5 MNaCl. The chloroform layer was shaken with 0.26% aqueous nickeloussulfate hexahydrate, dried over anhydrous sodium sulfate, and brought todryness in vacuum. The dry residue was dissolved in 2 ml hexane, andfiltered through GF/C grass fiber filter (Whatman). The hexane wasevaporated in vacuum to yield 0.334 g (66% of theory) of the product asa greyish-blue solid, readily soluble in hexane and chloroform. TLC: Rf0.16 (silica; CHCl₃—MeOH—H₂O 65:25:4). The intended structure wasconfirmed by PMR.

6-(Cholesteryl-succinyl)amino-2-(N,N-bis-carboxymethylamino)-hexanoicacid nickel salt (Chol-NTA-Ni) (IV).

244 mg of cholesteryl hemisuccinate (Sigma Chemical Co., USA) werereacted with N-hydroxysuccinimide and DCC, and further with compound Ihydrobromide by the same method as described for compound III. Uponaddition or the nickelous sulfate solution, a greenish paste formed. Thepaste was extracted several times with the chloroform-methanolmixture.(5:1 by vol.). The extract was dried over anhydrous sodiumsulfate, filtered through GF/C glass fiber filter, and brought todryness in vacuum. Yield 119.5 mg (30% of theory) of a greenish solid,readily soluble in chloroform giving greenish-blue solution. TLC: Rf0.12 (silica; CHCl₃—MeOH—H₂O 65:25:4). The intended structure wasconfirmed by PMR.

6-(1,2-Distearoyl-sn-glycerophosphoryl-ethanolaminocarbonyl)-poly(oxyethylene)-oxycarbonyl)amino-2-(N,N-bis-carboxymethylamino)-hexanoicacid nickel salt (DSPE-PEG-NTA-Ni) (V)

198 mg ( 0.0445 mmol) ofdistearoylphosphatidylethanolaminocarbonyl-poly(ethyleneglycol)-propionic acid N-hydroxysuccinimidyl ester (NHS-PEG-DSPE,Shearwater Polymers, Ala., USA) prepared from poly(ethylene glycol) withmol. weight 3,400 were dissolved in the mixture of 1 mL of anhydrousethanol and 0.5 ml of anhydrous chloroform, mixed with the solution of40.8 mg (0.120 mmol) of I hyrdobromide in 0.5 mL of anhydrous ethanoland 0.15 mL (1.08 mmol) of triethylamine, and stirred 2 hours at 60° C.The reaction mixture was brought to dryness and dissolved in 3 ml of0.14 M aqueous NaCl. The mixture was clarified by centrifugation at15,500×g for 5 min., and clear supernatant was brought to dryness invacuum. The residue was dissolved in 2.5 ml of 0.144 M NaCl, pH wasadjusted to 6.8 with 1 N NaCl, and 0.12 mL of 1 M NiSO₄ were added. Thesolution was chromatographed on a 13-mL column with cross-linked dextranbeads (Sephadex G-75, Pharmacia Amersham, USA) using 0.144 M NaCl aseluent. The fractions appearing at the void volume (total 4 mL) werecollected, and dried by lyophilization overnight. The lyophilized cakewas extracted with the mixture of 2 ml anhydrous ethanol and 0.2 mlchloroform; the insoluble matter was removed by centrifugation, and theclear solution was brought to dryness in vacuum. The residue wasredissolved in 2 ml of ethanol containing 0.1 ml of chloroform, thesolution clarified by centrifugation (15.5×g, 5 min), and brought todryness in vacuum. Yield 92 mg (46% of theory). The bluish solid wassoluble in chloroform-methanol mixture (60:40 by vol.) and in water,giving light-blue solutions. The intended structure was confirmed byPMR.

Formulation Into Liposomes.

Compounds III, IV, V in the amount of 0.5 mol. %, 1 mol. %, 2 mol. % or5 mol. % of the liposome lipid were formulated into small unilamellarliposomes prepared from POPC and cholesterol (3:2 molar ratio),containing fluorescent reporter HPTS, and tested in a CLIA assay usingHER2-overexpressing SKBR-3 cells and a recombinant anti-HER2 scFv F5with a hexahistidine tag as described in the Example 1 above. Theresults were similar to those described in Example 1 using DOGS-NTA-Ni.

Example 3 Intracellular Delivery of a Cytotoxic Liposome usingNi-NTA-PEG-DSPE and His-Tagged scFv Antibody

Liposomes having lipid composition of DSPC, cholesterol,methoxy-poly(ethylene glycol)-DSPE derivative (PEG(2000)-DSPE, PEG mol.weight 2,000; Avanti Polar Lipids, Alabama, USA), and compound V(Ni-NTA-PEG-DSPE) in the molar ratio of 3:2:0.05:0.06 were prepared bylipid film hydration and polycarbonate track-etched membrane (0.1 μm, 10times) extrusion at 55° C. in 0.25M aqueous ammonium sulfate. Afterremoval of unencapsulated ammonium sulfate and bringing the liposomesinto 5% dextrose, 5 mM morpholinoethanesulfonic acid (MES) buffer, pH5.5 (adjusted with sodium hydroxide) by gel-chromatography usingcross-linked dextran beads (Sephadex G-75, Pharmacia, New Jersey, USA),the liposomes were mixed with 10 mg/ml vinorelbine bitartrate solutionUSP (GlaxoWellcome, USA) to achieve drug/lipid molar ratio of 5:1 andincubated at 55° C. for 30 min. to achieve drug encapsulation.Unencapsulated vinorelbine was removed by gel-chromatography as above.Typically >80% of the drug remained encapsulated into so obtainedNi-NTA-PEG-DSPE-containing liposomes. Control liposomes were madesubstituting PEG-DSPE for Ni-NTA-PEG-DSPE, and were loaded withvinorelbine in a similar way. Liposomes containing covalently bound 4G7were prepared by incubating vinorelbine-loaded control liposomes with4G7 conjugated to an amphipathic linker, maleimido-PEG-DSPE(Papahadjopoulos, et al. U.S. Pat. No. 6,210,707). Bovine endothelialcells (BEND-3) expressing vascular endothelial growth factor (VEGF)receptor were incubated (37° C., 6 hours) in the growth mediumcontaining 0.03-90 microgram/mL of the free (i.e. non-encapsulated)vinorelbine, or vinorelbine encapsulated in the Ni-NTA-PEG-DSPEliposomes with or without 0.02 mg/mL of the internalizing anti-VEGFRscFv antibody 4G7 having a hexahistidine tag and a terminal cysteinegroup. The cells were post-incubated in the growth medium without thedrug for another 72 hours, and the viability of the cells was determinedby a conventional tetrasolium (MTT) assay. The median cytotoxic dose,i.e. the dose that reduces the cell viability to 50% of non-treatedcontrol (IC₅₀), was as follows: free vinorelbine, 0.67 μg/mL;vinorelbine in Ni-NTA-PEG-DSPE liposomes without 4G7 scFv, >100 μg/mL(IC₅₀ not reached); control liposomes+4G7 scFv, >100 μg/mL (IC₅₀ notreached); vinorelbine in liposomes with covalently bound 4G7, 2.5 μg/ml;Ni-NTA-PEG-DSPE liposomes+4G7 scFv, 1.4 μg/mL. Thus, we observedspecific delivery of vinorelbine into vascular epithelial cells byNi-NTA-PEG-DSPE-containing liposomes coupled to a receptor-specific scFvvia a hexahistidine tag.

Example 4 Targeted Delivery of Methotrexate into Cancer Cells byNi-NTA-PEG-DSPE Liposomes and a Hexahistidine-Tagged Antibody

Liposomes containing methotrexate were made from DSPC, cholesterol, andPEG(2000)-DSPE (molar ratio, 3:2:0.025) and 100 mg/mL solution ofmethotrexate sodium in 10 mM buffer solution ofN-4-hydroxyethyl-piperazino-ethylsulfonic acid (HEPES) sodium salt, pH7.2, by a reverse phase evaporation method of Szoka and Papahadjopoulos(Proc. Natl. Acad. Sci. USA, 75:4134-4178, 1978). Unencapsulatedmethotrexate was separated by gel-chromatography using 20 mM HEPES, 144mM NaCl (HBS buffer) as eluate. Resulting liposomes containing 150±7 mgmethotrexate per mmol of liposomal phospholipid, were incubated (55° C.,30 min) with Ni-NTA-PEG-DSPE, dissolved in HBS buffer, in the amount of2 mol. % of the liposomal phospholipid. No drug leakage from theliposomes was detected during this incubation. Median cytotoxic dose(IC₅₀) of these liposomes, with or without 4G7 scFv, as well as of thefree methotrexate, was determined in the culture of BEND3 cells asdescribed in the Example 3, to be as follows: free methotrexate, >90μg/ml (not reached); methotrexate in Ni-NTA-PEG-DSPE-liposomes withoutantibody, >90 μg/ml (not reached); methotrexate inNi-NTA-PEG-DSPE-liposomes in the presence of hexahistidine-tagged 4G7scFv, 9 μg/ml. Thus, incorporation of Ni-NTA-PEG-DSPE into pre-formedmethotrexate liposomes produced a methotrexate-carrying liposome thatformed an internalizable construct with a His-tagged antibody.

Example 5 Loading of Cytotoxic Drugs Into Liposomes Containing VariousNi-NTA Lipids

The nature of Ni-NTA lipid in the liposomes had an unexpected effect onthe efficiency of cytotoxin encapsulation by a transmembrane gradientmethod. Liposomes with entrapped 0.25 M ammonium sulfate were preparedusing the lipid matrix composed of DSPC, cholesterol (Chol), andPEG-DSPE in the molar ratio 3:2:0.03 (Preparation A), DSPC, Chol,PEG-DSPE, and Ni-NTA-DOGS in the molar ratio 3:2:0.03:0.06 (PreparationB), and DSPC, Chol, and Ni-NTA-PEG-DSPE (compound V) in the molar ratio3:2:0.03:0.06 (Preparation C). After bringing the liposomes into 5%dextrose, % mM MES-Na buffer pH 5.5 (MES-Dextrose), the liposomes wereincubated (55° C., 30 min) with vinorelbine (VNR) or doxorubicin (DOX)at the input drug/lipid ratio of 150 mg of the drug per mmol of theliposome phospholipid. The liposome were chilled in ice, and separatedfrom unencapsulated drug by gel-chromatography using MES-Dextrosebuffer. The concentration of liposome phospholipid was determinedspectrophotometrically by the molybdate-ascorbic acid method followingacid digestion of the liposomes. The concentration of the liposome drugwas determined spectrophotometrically after solubilization of theliposomes in 80% aqueous methanol (vinorelbine, absorbance at 370 nm),or 70% aqueous isopropanol-0.1M HCl (doxorubicine, absorbance at 485 nm)by comparison to standard curves. The loading efficiency was calculatedas percent encapsulated drug of total added for loading. The results areshown in Table 1. TABLE 1 Results for drug loading in liposomes. Drugloading, mg/mmol of Loading efficiency, Preparation Drug phospholipid %DSPC/Chol/PEG-DSPE (A) VNR 150.2 ± 4.9 100.2 ± 3.4 DSPC/Chol/PEG-DSPE/Ni- VNR  13.6 ± 1.2  9. ± 0.9 NTA-DOGS (B)DSPC/Chol/PEG-DSPE/Ni- VNR 149.6 ± 5.8 99.8 ± 4.0 NTA-PEG-DSPE (C)DSPC/Chol/PEG-DSPE (A) DOX 159.4 ± 6.1 106.3 ± 14.4DSPC/Chol/PEG-DSPE/Ni- DOX  40.4 ± 4.3 26.9 ± 3.0 NTA-DOGS (B)DSPC/Chol/PEG-DSPE/Ni- DOX  138.2 ± 10.2 92.2 ± 8.0 NTA-PEG-DSPE (C)

The nature of Ni-NTA-lipid had little effect on the encapsulation bydirect sequestration of the lipid hydration medium (HPTS, MTX), such asreverse phase evaporation. Direct sequestration, however, is inefficient(MTX loading efficiency 28.7-29.5%), compared to gradient methods thatprovide almost quantitative encapsulation. Thus, unexpectedly, only thepolymer-linked NTA lipid provided for the efficient loading of the drugsby an advantageous, transmembrane-gradient-based method.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-43. (canceled)
 44. A construct for screening a cell for aninternalizing receptor, said construct comprising a ligandnon-covalently coupled to an effector through an epitope tag.
 45. Theconstruct of claim 44, wherein said ligand is a peptide.
 46. Theconstruct of claim 44, wherein said ligand is selected from the groupconsisting of an scFv, an Fv, an Fab, a monoclonal antibody, a cytokine,an enzyme, a hormone, and a growth factor.
 47. The construct of claim44, wherein said ligand is a member of a combinatorial library.
 48. Themethod of claim 74, wherein said phage display library uses afilamentous phage.
 49. The construct of claim 44, wherein said epitopetag is selected from the group consisting of a His-tag, a Flag-tag, anHA-tag, a myc-tag, and a DYKDDDDK (SEQ ID NO:1) epitope.
 50. Theconstruct of claim 44, wherein the effector comprises a nanoparticle ora liposome.
 51. The construct of claim 44, wherein said epitope tag is ahexahistidine (His₆) tag and said effector is a liposome comprising anitrilotriacetic acid (NTA) lipid or an iminodioacetic acid (IDA) lipid.52. The construct of claim 44, wherein said ligand is an antobody andsaid epitope tag is attached to said antibody through a covalent linkageto protein A or protein G.
 53. The construct of claim 44, wherein saidconstruct is polyvalent for said ligand. 54-61. (canceled)
 62. A metalchelating lipid composition comprising a lipid, a hydrophilic polymer,and a metal chelation group attached to said hydrophilic polymer. 63.The metal chelating lipid composition of claim 62, wherein said metalchelation group is NTA or IDA.
 64. The metal chelating lipid of claim62, wherein said hydrophilic polymer comprises polynucleotide(ethyleneglycol).
 65. The metal chelating lipid of claim 62, wherein said lipidcomprises distearoylphosphatidylethanolamine (DSPE). 66-68. (canceled)69. A composition for delivering an agent to a cell, the compositioncomprising the construct of claim 44, wherein the effector comprises themetal chelation lipid composition of claim 62, wherein the metalchelation group of the metal chelation lipid composition is capable offorming a chelation bond with the epitope tag of the construct; a ligandcomprising said epitope tag where said ligand binds and is optionallyinternalized by a cell; and an agent associated with said metalchelation lipid composition.
 70. The composition of claim 69, whereinsaid metal chelation lipid composition comprises a liposome and saidliposome contains or is complexed with said agent.
 71. The compositionof claim 69, wherein said hydrophilic polymer of the metal chelationlipid composition comprised poly (ethylene glycol).
 72. The compositionof claim 69, wherein said lipid of the metal chelation lipid compositioncomprises distearoylphosphatidylethanolamine (DSPE).
 73. The compositionof claim 69, wherein the agent comprises a reporter, a cytotoxin, adrug, or a nucleic acid.
 74. The construct of claim 47, wherein saidcombinatorial library comprises a combinatorial chemical library, arecombinant library, or a phage display library.
 75. The construct ofclaim 44, wherein the effector comprises a reporter selected from thegroup consisting of an enzyme, a colorimetric label, a fluorescentlabel, a luminescent label, and a radioactive label.